Transformation questions
Mega
When I want to transform bacteria they have to be in exponential
growth.
So usually you'd have do do an over-night culture @ 37°C.
I don't have a breeding box, will they be in exponential growth also
at 21°C (but some 20 hours instead of just 12)???
Best regards,
Cathal Garvey
terrarium heater mat and a thermostat.
Also, after an overnight culture, the bacteria will usually be in
*stationary* phase, not exponential.
For exponential growth, try somewhere between 3 and 5 hours after
inoculation.
Personally, I've had success using the TSS method (PEG3350 Laxative and
Store-brand "Epsom Salt" Magnesium Chloride, no DMSO) by growing cells
at 30C, waiting for 3 hrs after inoculation before harvesting
exponential cells for transformation. I used overnight-grown cells to
inoculate fresh culture that morning, so the cells weren't that
exhausted. I used a centrifuge to concentrate cells before
transformation, but you don't have to; you can directly dilute growing
cells with 2x TSS transformation medium, instead.
When growing cells without a shaker, try to grow them at the bottom of a
wide-bottomed flask in a thin layer of broth, 3-5mms deep, so that
oxygen can penetrate. Give them an occasional stir/shake to break up
biofilms and encourage even growth.
--
www.indiebiotech.com
twitter.com/onetruecathal
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PGP Public Key: http://bit.ly/CathalGKey
Mega
For just three hours I could also use a water bath of which I check
the temperature say all 15 min... Or a hand-warmer...
Mega
I got 3 Malt-extract-agar petri dishes that are ready to use.
The diameter is 95 mm and height of the agar is approximately 5mm.
So I calculated to have around 140ml of agar in the petri dish.
Now I have to get ampicillin, dilute it in water. Then spread the amp-
water over the plate to make it an amp-agar.
100ug/ml amp ws mentioned. So I would need around 15 mg of ampicillin.
Shall I better take 20 to be sure it works??
Jeswin
> Another question:
>
> I got 3 Malt-extract-agar petri dishes that are ready to use.
>
> The diameter is 95 mm and height of the agar is approximately 5mm.
> So I calculated to have around 140ml of agar in the petri dish.
>
> Now I have to get ampicillin, dilute it in water. Then spread the amp-
> water over the plate to make it an amp-agar.
>
I'm not fully sure but why not add the amp into the bacteria you're
going to plate? Is this a bad idea?
> 100ug/ml amp ws mentioned. So I would need around 15 mg of ampicillin.
> Shall I better take 20 to be sure it works??
>
Sounds like a lot of amp. The ampicillin I use is 1:1000 dilution of
100mg/mL stock solution, so its 1 uL per 1 mL that I commonly use. I
might be wrong though.
Mega
going to plate? Is this a bad idea?
resistance... Else all of them - also the transformants - would be
destroyed...
20 mg solid amp is too much??
On 24 Jan., 17:36, Jeswin <phillyj...@gmail.com> wrote:
Cathal Garvey
Nathan McCorkle
>> water over the plate to make it an amp-agar.
>>
> I'm not fully sure but why not add the amp into the bacteria you're
> going to plate? Is this a bad idea?
>
>> 100ug/ml amp ws mentioned. So I would need around 15 mg of ampicillin.
>> Shall I better take 20 to be sure it works??
>>
Mega, it looks like you have good math, but the difference is 100ug/mL
is for ampicillin when its MIXED into the agar... you're talking about
spreading it on TOP. While it will diffuse down into the agar, it will
take a while, probably won't be 100% diffused (but you only care about
the top surface really, but you won't know EXACTLY what concentration
of drug you have at the top because its not completely mixed)
A quick google for "spread spreader ampicillin" brought this up:
http://www.protocol-online.org/biology-forums/posts/36393.html
where the outcome is, you can spread ampicillin on top, but then you
should let the plates dry for a few hours in an incubator (if you had
a laminar flow hood I would dry with the top of the plate open until
you could see the excess water dried up)... they say to let them dry
inverted so you don't get condensation that drips on to the agar
surface (will splash your ampicillin around, and if you HAD put any
bacteria, they could also get splashed and moved around, so always
incubate inverted!)
> Sounds like a lot of amp. The ampicillin I use is 1:1000 dilution of
> 100mg/mL stock solution, so its 1 uL per 1 mL that I commonly use. I
> might be wrong though.
>
Jeswin, if you had plate with 100mL agar, you'd be using 10mg of
ampicillin (1uL/1mL agar, 100mL agar == 100uL amp juice, 100uL==0.1mL,
100mg/mL 100*0.1==10mg)
> --
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--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Jeswin
> Jeswin, if you had plate with 100mL agar, you'd be using 10mg of
> ampicillin (1uL/1mL agar, 100mL agar == 100uL amp juice, 100uL==0.1mL,
> 100mg/mL 100*0.1==10mg)
>
>> --
estimating things.
By the way, Mega, what is the project you are working on?
Mega
It'll be my first project to engineer something genetically.
I now just have to get ampicillin.... In the drug store they don't
have it on store and technically you would need a prescription for
it.
I saw that there is some medicine for fish (aquarium) made of amp...
But it will cost more than 10€ to ship and some 20 €for 4 packs of
7g ... (I just need 20 mg)
Jonathan Nesser
Tom Randall
On Jan 25, 8:43 pm, Jonathan Nesser <jonathan.nes...@gmail.com> wrote:
> I was wondering about that, what are the legal issues regarding obtaining
> ampicillin or other DEA scheduled antibiotics for research use? I found a
> site that sold small amounts of ampicillin (didn't inquire as I'm trying to
> stay away from antibiotics per Cathal's argument of environmental
> implications), but it would be nice to know. I was also wondering about
> horomones/amines like norepinephrine, dopamine, etc.
Try
http://www.bostonbioproducts.com
Catalog # P-740, 25 g $80.00
www.goldbio.com
Catalog # A-301-25
25 g for $39.00, other quantities available, also a lot of other
common antibiotics,
also deliver to residential addresses like boston bioproducts.
www.chemsavers.com has it, but more expensive, also delivers to
residential
I am sure there are others. No environmental issues with amp or others
if you autoclave everything before you dispose of it, this will
inactivate most antibiotics that I am aware of. If there are any that
can withstand autoclaving I would be interested in knowing. This is
why one normally adds antibiotics to media after autoclaving the media
and cooling to ~55-60C to avoid their inactivation.
Go wild!
Venkatesh Srinivas
> ...
> I am sure there are others. No environmental issues with amp or others
> if you autoclave everything before you dispose of it, this will
> inactivate most antibiotics that I am aware of. If there are any that
> can withstand autoclaving I would be interested in knowing. This is
> why one normally adds antibiotics to media after autoclaving the media
> and cooling to ~55-60C to avoid their inactivation.
> Go wild!
I believe chloramphenicol is thermostable; should survive autoclaving.
Not commonly used in DIYbio I imagine though...
--vs;
Mega
http://www.ebay.at/itm/Ampicillin-7-5g-sachets-for-Birds-Pigeons-4-Pcs-/280798961653?pt=UK_Collectables_AnimalCollectables_SM&hash=item4160ec1ff5
It will cost below 15 € (included shipping), athought it's very
much... ( I could need only one of them)
I think this is the only chep way to get it without prescription...
On 26 Jan., 05:05, Tom Randall <tarand...@gmail.com> wrote:
> On Jan 25, 8:43 pm, Jonathan Nesser <jonathan.nes...@gmail.com> wrote:
>
> > I was wondering about that, what are the legal issues regarding obtaining
> > ampicillin or other DEA scheduled antibiotics for research use? I found a
> > site that sold small amounts of ampicillin (didn't inquire as I'm trying to
> > stay away from antibiotics per Cathal's argument of environmental
> > implications), but it would be nice to know. I was also wondering about
> > horomones/amines like norepinephrine, dopamine, etc.
>
> No real legal issues in the US at least.
>
> Catalog # P-740, 25 g $80.00
>
> www.goldbio.com
> Catalog # A-301-25
> 25 g for $39.00, other quantities available, also a lot of other
> common antibiotics,
> also deliver to residential addresses like boston bioproducts.
>
Nathan McCorkle
Mac Cowell
Look around online for those filters and syringe pistons so you can sterilize your amp. Does the bird variety come in pill form, or paste, or powder, or what?
231.313.9062 // @100ideas // sent from my rotary phone
Mega
that.... although the pills are packaged one by one seperately. So it
would be out of reach from oxygen and germs....
On 26 Jan., 19:18, Mac Cowell <m...@diybio.org> wrote:
> I've used small volumes of GoldBios amp. In the smaller sizes they send along a handy plastic syringe with a screw-on 2-micron filter. So you just mix the amp powder with water and squirt it through the filter to sterilize it.
>
> Look around online for those filters and syringe pistons so you can sterilize your amp. Does the bird variety come in pill form, or paste, or powder, or what?
>
> 231.313.9062 // @100ideas // sent from my rotary phone
>
> On Jan 26, 2012, at 6:23 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
>
>
>
> > That should do the trick. Its cheap too, you won't have to buy for a while!
>
> >> Shipment to Europe will be expensive... What about that:
>
> >> It will cost below 15 € (included shipping), athought it's very
> >> much... ( I could need only one of them)
>
> >> I think this is the only chep way to get it without prescription...
>
> >> On 26 Jan., 05:05, Tom Randall <tarand...@gmail.com> wrote:
> >>> On Jan 25, 8:43 pm, Jonathan Nesser <jonathan.nes...@gmail.com> wrote:
>
> >>>> I was wondering about that, what are the legal issues regarding obtaining
> >>>> ampicillin or other DEA scheduled antibiotics for research use? I found a
> >>>> site that sold small amounts of ampicillin (didn't inquire as I'm trying to
> >>>> stay away from antibiotics per Cathal's argument of environmental
> >>>> implications), but it would be nice to know. I was also wondering about
> >>>> horomones/amines like norepinephrine, dopamine, etc.
>
> >>> No real legal issues in the US at least.
>
> >>> Tryhttp://www.bostonbioproducts.com
>
> >>> Catalog # P-740, 25 g $80.00
>
> >>>www.goldbio.com
> >>> Catalog # A-301-25
> >>> 25 g for $39.00, other quantities available, also a lot of other
> >>> common antibiotics,
> >>> also deliver to residential addresses like boston bioproducts.
>
>
> >>> I am sure there are others. No environmental issues with amp or others
> >>> if you autoclave everything before you dispose of it, this will
> >>> inactivate most antibiotics that I am aware of. If there are any that
> >>> can withstand autoclaving I would be interested in knowing. This is
> >>> why one normally adds antibiotics to media after autoclaving the media
> >>> and cooling to ~55-60C to avoid their inactivation.
> >>> Go wild!
>
> >> --
> >> You received this message because you are subscribed to the Google Groups "DIYbio" group.
> >> To post to this group, send email to diy...@googlegroups.com.
> >> To unsubscribe from this group, send email to diybio+un...@googlegroups.com.
Cathal Garvey
Venkatesh Srinivas <m...@endeavour.zapto.org> wrote:
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Mega
documentation-infotainment' which costed about 20 bucks and they
tested if it really worked.
And, surprisingly, it really worked fine!
Maybe Microwave sterilization could also work when it damages the DNA
( not only heat up the bacteria). Or you add e.g. ampicillin to kill
the bacteria and then heat the whole pot to destroy the amp.
On 26 Jan., 23:17, Cathal Garvey <cathalgar...@gmail.com> wrote:
> Actually, a control Bacillus plasmid I'm going to be using is Chloramphenicol resistant: I hadn't realised it was heat resistant! I'd better imagine up a disposal procedure..
>
>
>
>
>
>
>
>
>
> Venkatesh Srinivas <m...@endeavour.zapto.org> wrote:
Mega
Cathal Garvey
antibiotics present; it's only unused media that really needs boiling or
other such treatment before disposal. In the case of chloramphenicol,
I'll probably just add some resistant cells to unused media and incubate
in order to destroy the broth, then boil-kill and UV inactivate.
Microwaves don't explicitly damage DNA.
However, UV does, and I would advocate using UV to mince DNA before
disposal, especially if the DNA contains medically significant (i.e.
AmpR, maybe Chlor?) antibiotic resistance genes. Most labs have UV
transilluminators for Gel electrophoresis, even though blue illumination
is more all-round useful (precisely because it doesn't mince DNA).
So, if I were working with E.coli, which can't survive boiling, I'd
simply boil cellular waste in a glass beaker or flask, then put the
container on a UV illuminator for 5 minutes before disposal.
If I were working with B.subtilis, I'd autoclave rather than boiling,
and do the same.
Clearly, I wouldn't be so strict with DNA that was proven to have no
ecological consequences; no resistance genes, no ecological-unknowns. A
plasmid containing only GFP and plasmid maintenance genes is hardly
worth worrying about, for example.
Of course, opinions differ, and we've had animated discussions here in
the past on this issue; whether to bother destroying antibiotics,
whether to bother destroying DNA.
Mega
ecological consequences; no resistance genes, no ecological-unknowns.
A
plasmid containing only GFP and plasmid maintenance genes is hardly
worth worrying about, for example. "
By the way, the gfp could not be taken up and expressed by plant
seemen, because the (bacterial) promotor wouldn't be readable?? It
would be junk - DNA?
Mega
To transform bacteria they have to be in a state of exponential
growth. Is this really imperative or just increases transformation
efficiency??
____
For my transformation I think about building a breeding box using an
ATtiny, LM335, a box of polystyrol and some wire.
But in case this fails, would transformation still work?
Nathan McCorkle
using a cell-phone vibrator (or playstation/nintendo controller
vibrator) attached to an eppendorf tube for shaking/aeration on the
cheap
--
Cathal Garvey
broth. You should still give them an occasional swirl though.
And yes, they should be warm. I have successfully transformed E.coli
grown at 30C. I incubate using a terrarium heater mat, a pet-shop
thermostat, and a polystyrene box. I use an external thermometer to
sanity-check the temperature, because commercial pet thermostats are
rarely correct (but very stable, I find).
If using heater wire to heat your terrarium, be careful to insulate the
wire somehow to keep it from melting/igniting the polystyrene!
Mega
calcium chlorate but chlorine.
I googled it up and it's used in homoeo-pathy... But is it then pure
calcium chlorine? Or has it a homeopathic dosage (1 molecule cacl2 in
1g of the salt?) ? So it would be of no sense for transformation.
I also made my own cacl2 from egg peal with HCl.... But I fear there
are proteins soluted (I should have burnt them before)
On 31 Jan., 10:25, Cathal Garvey <cathalgar...@gmail.com> wrote:
> You can get away without shaking, *if* the cultures are grown in shallow
> broth. You should still give them an occasional swirl though.
>
> And yes, they should be warm. I have successfully transformed E.coli
> grown at 30C. I incubate using a terrarium heater mat, a pet-shop
> thermostat, and a polystyrene box. I use an external thermometer to
> sanity-check the temperature, because commercial pet thermostats are
> rarely correct (but very stable, I find).
>
> If using heater wire to heat your terrarium, be careful to insulate the
> wire somehow to keep it from melting/igniting the polystyrene!
>
> On 31/01/12 09:16, Nathan McCorkle wrote:
>
>
>
>
>
>
>
>
>
> > they really should be warm and shaking before transformation... try
> > using a cell-phone vibrator (or playstation/nintendo controller
> > vibrator) attached to an eppendorf tube for shaking/aeration on the
> > cheap
>
Anselm Levskaya
Ampicillin - most definitely add it to the agar, you want a uniform
concentration of the stuff. Top spreading takes forever. Just add
the amp to your agar mix after it's cooled down to merely "hot" and
not boiling.
Amp is decent at selecting from anywhere around 2ug/mL up to
1000ug/mL, we typically use 50-100ug/mL in the lab. A light "dusting"
per plate by eye is generally enough. 100mg/L will work fine as a
recipe.
Amp is very convenient in that you don't have to wait for the
resistance gene to be expressed before adding it. Since it attacks
cell wall growth and not translation, you can just add it. With
kanamycin, chloramphenicol, etc. you have to wait an hour.
---
Re competent cells: I agree that the TSS competency method is
probably the easiest to get to work in a home lab setting.
electrocompetent preps are even easier but you'd then need to
build/obtain a 2500V exponential wave shocker. (Highly recommend
electrocompetent methods if you want really high competency, i.e. if
you ever want to try a random library screen / selection.)
It is absolutely essential that you use "midlog" exponentially growing
cells for the competency prep. You want the cells rapidly dividing so
that their cell wall hasn't yet fully thickened, as it does once you
get to stationary phase. You'll also probably want to keep the
cultures shaking for the exponential growth phase if you want
highly-competent cells. As soon as you stop their growth keep the
cells rigorously cold at 4deg for the best competency.
-A
Nathan McCorkle
> Tips:
>
> Ampicillin - most definitely add it to the agar, you want a uniform
> concentration of the stuff. Top spreading takes forever. Just add
> the amp to your agar mix after it's cooled down to merely "hot" and
> not boiling.
>
> Amp is decent at selecting from anywhere around 2ug/mL up to
> 1000ug/mL, we typically use 50-100ug/mL in the lab. A light "dusting"
> per plate by eye is generally enough. 100mg/L will work fine as a
> recipe.
>
> Amp is very convenient in that you don't have to wait for the
> resistance gene to be expressed before adding it. Since it attacks
> cell wall growth and not translation, you can just add it. With
> kanamycin, chloramphenicol, etc. you have to wait an hour.
>
> ---
> Re competent cells: I agree that the TSS competency method is
> probably the easiest to get to work in a home lab setting.
> electrocompetent preps are even easier but you'd then need to
> build/obtain a 2500V exponential wave shocker. (Highly recommend
> electrocompetent methods if you want really high competency, i.e. if
> you ever want to try a random library screen / selection.)
Mega this is pretty good info, you should try using a piezo electric
sparker, commonly found in electronic ignition household butane
lighters (the long grill lighters generally have them).
Read the two posts here with info compiled by Simon Quellen Field
about how to use a piezo sparker and a potentiometer to adjust the
voltage, which for E.coli is generally 18kV/cm (or 1.8kV if the
electrodes in the cuvette are 0.1cm apart).
I successfully electroporated mid-log phase (phase is important as
Anselm said) E.coli with no preparation to the cells other than 2
rinses with distilled and filtered water (filtered of ions such that
the resistance of the water is about 18 Mega Ohms) (though distilled
and sterile should be just fine).
I did use a commercial electroporator, and commercial cuvettes (you
have to be careful of the metal used for the electrodes if you make
your own reactor, use gold or platinum, because they are pretty
non-reactive metals if they come into solution from the spark)
Mega
What I have planned so far:
Do a calcium chloride transformation and one PEG transformation and
see what works best under garage conditions.
DIY electroporation also sounds great (do the cells in the
electroporator have to be in exponential growth?)
For the electrodes I might try graphit, from an old battery (as my
former chemistry teacher recommended for making electrolysis - does
not corrode )
On 5 Feb., 08:40, Nathan McCorkle <nmz...@gmail.com> wrote:
Nathan McCorkle
mid-log/exponential phase. This is important because they're in a very
healthy state nutrient-wise, and as Anselm pointed out their cell
walls may be thinner (I've never heard that mentioned before, but I'm
inclined to say it sounds reasonable)
Graphite sounds like decent electrodes, only one way to see if it works!
> --
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Mega
I thought that the electricity can go through the cell wall anyway if
it's strong enough.
And exponential growth with thin cell walls is better for chemical
transformation.
Mega
Well, for the cacl2 transformation - whatconcentration will I need???
I read 0.1 molar, 0.05molar, 0.07 molar??
What suits best??
Nathan McCorkle
> For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
Mega
If you have a look at page 5 of the pdf (page 2 titled below) at the
very bottom of the page, you read ~ 'if you have no incubator, just
put it 48 hours to 72 hours at ambient room temperature'
> broth. You should still give them an occasional swirl though.
>
> And yes, they should be warm. I have successfully transformed E.coli
> grown at 30C. I incubate using a terrarium heater mat, a pet-shop
> thermostat, and a polystyrene box. I use an external thermometer to
> sanity-check the temperature, because commercial pet thermostats are
> rarely correct (but very stable, I find).
>
> If using heater wire to heat your terrarium, be careful to insulate the
> wire somehow to keep it from melting/igniting the polystyrene!
>
> On 31/01/12 09:16, Nathan McCorkle wrote:
>
>
>
>
>
>
>
>
>
> > they really should be warm and shaking before transformation... try
> > using a cell-phone vibrator (or playstation/nintendo controller
> > vibrator) attached to an eppendorf tube for shaking/aeration on the
> > cheap
>
Mega
Next tuesday I'll try CaCl2 transformation. Then I count the
colonies.
Thereafter I'll conduct PEG transformation and count again the number
colonies.
Nathan McCorkle
> http://www.bio-rad.com/LifeScience/pdf/Bulletin_9563.pdf
>
> If you have a look at page 5 of the pdf (page 2 titled below) at the
> very bottom of the page, you read ~ 'if you have no incubator, just
> put it 48 hours to 72 hours at ambient room temperature'
Looks good! I suspect your efficiency really goes down without the
mid-log/exponential phase cells, but you're only trying to get one
plasmid in, so you really don't need good efficiency, as long as 1
cell gets the DNA and can replicate on the antibiotic plate!
Jeswin
have no colonies or just 1 or 2. Yesterday, the boss saw that the
water bath was at 41(maybe between 40 and 41 also) degrees instead of
42. Does that 1 degree make such a big difference in transformation
efficiency? Previously, I had gotten about 8 good colonies and a few
double colonies.
Cathal Garvey
share my thoughts:
With only seven degrees' difference between optimum growth and onset of
heatshock, I imagine a two degree difference (i.e. 40C) makes all the
difference, really. This may be more due to the decreased penetration of
heat energy due to a smaller thermal gradient than any difference to the
E.coli; after all, heat shock response is heat shock response, and it
probably sets in around 39C. But still; one way or another, it probably
does make a little difference.
However, I wouldn't expect tenfold reductions in efficiency over a
degree or so of difference; if you were expecting 80 colonies and only
got 8, is it possible that your DNA is toxic? Has the expression system
or whatever you're using been tested previously in E.coli? Gene toxicity
is a real thing; sometimes the most innocuous seeming genes can be toxic
when introduced into another species. With several hundred thousand
different molecules whizzing around inside a cell, it's not surprising
that sometimes you'll just get an incompatibility cropping up.
Also, if efficiency isn't a concern, the PEG/Mg method really does take
less time and effort. It's comparably efficient to a regular CaCl
transformation, but I don't know if you can push it up to the ideal CaCl
transformation frequencies used for libraries. But still; it works great
for routine transfections, and takes less time, thought and effort to
do. And no water baths.
Nathan McCorkle
(Bacterial Alkaline Phosphatase). If you did use BAP or similar, I'd
look at your ligation results on a gel to see if that was actually
successful.
1 or 2 or 8 colonies is really low for a transformation regardless of
the protocol. In my experience and reading, the heat shock temperature
and time doesn't really matter for pass/fail, those are knobs you turn
for tuning efficiency with a particular host strain.
I would look at the rest of your experiment, did you pre-chill all
your pipette tips, CaCl2 rinse solution, eppendorf tubes and other
consumables that were involved with the E.coli in a freezer for an
hour or so? Were you sure to be gentle with the cold E.coli to avoid
shear stress? Were you sure to go directly from an ice bath to heat
shock and back to an ice bath? Quick temperature swings are likely to
be a more important factor, you need the thermal gradient to be as
strong as possible, which means taking the ice bucket with your
chilled cells+DNA over to the water bath and moving as fast as you
can.
Are you recovering with SOC media, or just LB? Are you shaker
incubating for an hour before adding selective pressure?
Jeswin
> Jeswin, was this post-ligation? If so did you use BAP or similar
> (Bacterial Alkaline Phosphatase). If you did use BAP or similar, I'd
> look at your ligation results on a gel to see if that was actually
> successful.
>
> I would look at the rest of your experiment, did you pre-chill all
> your pipette tips, CaCl2 rinse solution, eppendorf tubes and other
> consumables that were involved with the E.coli in a freezer for an
I had my competent cells thaw on ice. I put my tips and tubes on ice,
but not 1 hour; more like 5 to 10 minutes. After mixing gently (finger
flick lightly), I left them on ice 30 minutes.
> hour or so? Were you sure to be gentle with the cold E.coli to avoid
> shear stress? Were you sure to go directly from an ice bath to heat
> shock and back to an ice bath? Quick temperature swings are likely to
I tried to be very careful with the cells, never letting them off the
ice more than a quick second to pipet them out. Ice to bath to ice,
less than a second in the air. Then I incubate for 45 minutes after
which I plated them.
> be a more important factor, you need the thermal gradient to be as
> strong as possible, which means taking the ice bucket with your
> chilled cells+DNA over to the water bath and moving as fast as you
> can.
>
> Are you recovering with SOC media, or just LB? Are you shaker
> incubating for an hour before adding selective pressure?
After the 45 minutes in shaker incubator at 37 degrees, I added 0.3 mL
LB. What is selective pressure?
Nathan McCorkle
> On Thu, Feb 9, 2012 at 12:40 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
>> Jeswin, was this post-ligation? If so did you use BAP or similar
>> (Bacterial Alkaline Phosphatase). If you did use BAP or similar, I'd
>> look at your ligation results on a gel to see if that was actually
>> successful.
>>
> No BAP
Did you just get done with a ligation though?
>
>> I would look at the rest of your experiment, did you pre-chill all
>> your pipette tips, CaCl2 rinse solution, eppendorf tubes and other
>> consumables that were involved with the E.coli in a freezer for an
>
> I had my competent cells thaw on ice. I put my tips and tubes on ice,
> but not 1 hour; more like 5 to 10 minutes. After mixing gently (finger
> flick lightly), I left them on ice 30 minutes.
>
>> hour or so? Were you sure to be gentle with the cold E.coli to avoid
>> shear stress? Were you sure to go directly from an ice bath to heat
>> shock and back to an ice bath? Quick temperature swings are likely to
>
> I tried to be very careful with the cells, never letting them off the
> ice more than a quick second to pipet them out. Ice to bath to ice,
> less than a second in the air. Then I incubate for 45 minutes after
> which I plated them.
>
>> be a more important factor, you need the thermal gradient to be as
>> strong as possible, which means taking the ice bucket with your
>> chilled cells+DNA over to the water bath and moving as fast as you
>> can.
>>
>> Are you recovering with SOC media, or just LB? Are you shaker
>> incubating for an hour before adding selective pressure?
>
> After the 45 minutes in shaker incubator at 37 degrees, I added 0.3 mL
Wait, you added the recovery media AFTER incubating? You're supposed
to do that (immediately or 2 minutes, can't remember which) after the
heat shock and transfer to ice again.
> LB. What is selective pressure?
antibiotics, etc...
Nathan McCorkle
Overview of CaCl2 Transformation Protocol:
Pre-chill these reagents - CaCl2 solution (20-50mM), E. coli,
plasmids, (4) 15mL orange-capped tube, (3) fresh 1mL tubes
add 15ml E.coli to orange-capped tube, spin at 2700g for 10 mins
decant supernatant, a little leftover is OK
add CaCl2 solution (5-30mL)
chill up to 20 mins
spin at 2700g for 10 mins, decant supernatant, resuspend in 1mL CaCl2
chill up to 20 mins
transfer 200uL of E.coli to each of (3) fresh 15mL orange-capped tubes
spin plasmids 15-30 seconds, transfer 10uL of each to a respectively
labeled tube
chill 20-30 mins
heat shock for 45-90 seconds, then immediately transfer to ice
after 1-2 mins, add 600-800uL LB broth (SOC is better)
place in 37 degree C incubator (can be shaking but at less than 50
RPM) for 45-60 mins
plate each transformant culture on appropriate antibiotic selection media
(originally posted here:
http://groups.google.com/group/diybio/browse_thread/thread/fa9fd3c4df71d8d0#msg_e609ce6a73333d5e
)
Jeswin
>
> Did you just get done with a ligation though?
>
> Wait, you added the recovery media AFTER incubating? You're supposed
> to do that (immediately or 2 minutes, can't remember which) after the
> heat shock and transfer to ice again.
>
My mistake, I read my notes wrong. I did add the LB after the 2
minutes on ice after heat shock
Mega
When I keep the cells on ice for 30 minutes, do I keep them litterally
on crashed ice or make a water bath which contains crushed ice??
I think a wather bath would be better because the liquid cools the
tubes better than ice cubes (between them is warm air)
Jeswin
solid surface rather than floating. Not sure if it really matters or
is only for convinience.
Nathan McCorkle
the lid of the test tube
On Mon, Feb 13, 2012 at 12:47 PM, Mega <masters...@gmail.com> wrote:
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Mega
I incubated them fpr 4 hours. Then I noticed that only the water was
32,2°C, the petri dish had 21°C !!
Now I made it that it has 31 - 32°C (by insulating the top)
You think 2 or 2.5 hours @ 32°C would be enough????
On 14 Feb., 01:15, Nathan McCorkle <nmz...@gmail.com> wrote:
> Just use crushed ice, you'll risk contamination if water gets around
> the lid of the test tube
>
> > Important question for my transformation tomorrow:
>
> > When I keep the cells on ice for 30 minutes, do I keep them litterally
> > on crashed ice or make a water bath which contains crushed ice??
>
> > I think a wather bath would be better because the liquid cools the
> > tubes better than ice cubes (between them is warm air)
>
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Mega
Firstly I made them grow exponentially, then put them into a microtube
containing some 250 ul 0.05m CaCl2. Then put them on ice for 30 mins.
After 30 min I noticed 'I've forgotten the plasmids' . S**t... then
added the plasmids and put it on ice for another 20 mins...
Then heatshock (42°C @ 43 sek). Immediately placed again on ice. 2
min. Then waited some 20-30 min for expression. Then put on amp-Agar.
Incubating them at room temperature...
Tomorrow (after 16 hours) see if it has worked.
Nathan McCorkle
> Ok. ready - transformation done.
>
> Firstly I made them grow exponentially, then put them into a microtube
> containing some 250 ul 0.05m CaCl2. Then put them on ice for 30 mins.
> After 30 min I noticed 'I've forgotten the plasmids' . S**t... then
> added the plasmids and put it on ice for another 20 mins...
>
> Then heatshock (42°C @ 43 sek). Immediately placed again on ice. 2
> min. Then waited some 20-30 min for expression. Then put on amp-Agar.
>
Did you give it a mL or 2 of media after the heatshock then 2 minutes on ice?
> For more options, visit this group at http://groups.google.com/group/diybio?hl=en.
Mega
>
> Did you give it a mL or 2 of media after the heatshock then 2 minutes on ice?
diluted honey or dog food or flour...
But finally I went on without.
Nathan McCorkle
media addition after the heatshock and 2min on ice is critical.
Mega
> >Amp is very convenient in that you don't have to wait for the
resistance gene to be expressed before adding it. Since it attacks
cell wall growth and not translation, you can just add it. With
kanamycin, chloramphenicol, etc. you have to wait an hour.
Mega
Nathan McCorkle
plate! I've always had the cells growing in liquid media first!
Do you have a centrifuge?
Mega
> Do you have a centrifuge?
Cathal Garvey
both methods probably require liquid media to work well.
You can make a form of LB cheaply at home using
digestive-aid-tablets/meat tenderiser (1tsp) to digest soy protein
(dissolve both in water, leave to work for a few hours), and adding
yeast extract and salt. Then filter coarsely, filter finely, filter
until clear. Then add agar if desired, or leave out for broth, and
sterilise by pressure cooker.
The recipe for LB (Lysogeny Broth) is:
10g digested protein (usually casein, soy in this case)
5g Yeast Extract (No-added-salt, or balance by adding less salt below)
2/5/10g salt, depending on who you ask.
Technically my way is closer to "tryptic soy broth" because it uses soy
protein rather than casein. I don't think it's worth trying to make
casein hydrolysate (AKA "Tryptone") at home, because most off-the-shelf
"milk protein" will contain a lot of lactose, which will interfere with
IPTG-inducible gene cassettes. Easier to avoid entirely and go with soy
protein.
Dakota Hamill
I am eager to see/hear of the results! Keep us posted
Mega
Not one colony...
But it's not over as long as I have some plasmids left.
Men, I really should have done a control culture to see if they
survived the heat shock...
It was clear to me that they would...
Possible errors:
Too much cold (I was told it can be too cold before heat shock)
Not adding LB to the heat shocked bugs...
Too much amp (a friend of mine asked) ??
Nathan McCorkle
Mega
I've had the petri dishes in the basement (Some 3-5°C). They should
survive this (but not grow)
Then they were 21°C for 4 hours and then 32-33°C for another 5 hours?
Cathal Garvey
cells for transformation, it's just not going to work right for a
first-time experiment.
Keep the DNA until you can do a transformation with liquid media. As
before, I suggest PEG/Mg for a beginner, but if you're doing CaCl, you
*have* to do it right, or it won't work.
Remember, CaCl isn't about *adding* CaCl; it's about washing away the
original medium using a centrifuge, and repeatedly re-suspending the
bacteria in CaCl at 4C. It's boring and time-consuming, but if you just
add CaCl and DNA and heat-shock, nothing will happen.
In contrast, if you just add cold (4C) LB with 20% PEG-3350 and 10g/L
MgSO4 to chilled exponential-phase cells, and add DNA, and leave on ice
for 30 minutes, something will probably happen. But again, it *has* to
be liquid broth for this to work well.
Nathan McCorkle
do you need to digest soy/casein supplements, or are short enough
peptide chains out of the box (I'm talking off the food store shelf).
I imagine some brands would claim to be 'gentler' on the protein
extraction, etc...
Cathal Garvey
it's a crude protein extract, probably containing tons of impurities.
Hence my habit of avoiding milk protein supplements in favour of soy.
I digest the soy with bromelain rather than trypsin, and I add an excess
of bromelain to overcome the natural trypsin inhibitors* in soy protein.
To calculate the excess, read how they calculate the strength of the
bromelain; it's usually listed in "Gelatin-digesting units", where a
unit can prevent X grams of gelatin from polymerising. I'd double the
dose needed to digest the weight of soy protein you're working with; if
1g bromelain digests 10g gelatin, use 2g bromelain to digest 10g soy
protein.
It's not scientific, but my E.coli cells grow on it, so I don't much
care right now. When I get into more specific requirements for
high-yield or precisely replicable growth, that's another matter, but
right now I use E.coli as a vector-carrier and little else. :)
*Trypsin inhibitors probably inhibit the function of the active site, a
triad of catalytic amino acids that are shared AFAIK with bromelain; so,
I assume they inhibit bromelain, also.
Mega
http://www.amazon.com/Miralax-30-Day/dp/B002FRQIQQ
This product would fit well?? Should contain PEG 3350.
Mega
http://www.amazon.com/Polyethylene-Solution-Laxative-Original-Prescription-4-1-Ounce/dp/B001H54UH6
Should be the same? But the title says it contains PEG-3350.
Mega
It said:
Product price: 5,9€
Shipping and handling: Wait for it.... 27€!!
That made me angry... So I called the pharmacist if they could get
one. In Austria and Germany they have none on stock.... They'll call
their delivery company and tell me...
On 16 Feb., 09:01, Mega <masterstorm...@gmail.com> wrote:
> Found this:http://www.amazon.com/Polyethylene-Solution-Laxative-Original-Prescri...
Nathan McCorkle
have Carrefour/Wal-Mart equivalent (do people outside the U.S. know
what Wal-Mart is???), if not I would visit in-person your local
pharmacies and grocery store pharmacy sections. Maybe some/most
employees don't know the ingredients for non-prescription items???
Mega
no idea what that may be.
But I'll look at some pharmacist.
(also on ebay, 6-15€ for the product. ok. but 19€ for the shipping!!)
Cathal Garvey
Mega
What I have:
transformation agents:
MgSO4
LB Agar dishes
Ice
(PEG missing)
selection:
Ampicillin
main actors:
E.Coli
pVIB
instruments:
adjustable pipette
incubator 32-33°C
Is that enough??
LB medium in liquid state is essential?
When I make my own, the bromelain comes from the pharmacist? Because
my internet paradoxon (find it cheap, but shipment costs are extreme).
Has anyone done research on using pinapple 'juice' from fresh
pinapples to digest it?? There should be one 'DIY Bio store' where I
get all stuff from, so I would have to pay the shipment only once...
Are there alternatives? What about a dirty mix of honey, pinapple and
milk, sterilized? Milk+Pineapples -> proteins, honey -> fructose,
glucose.
Flour diluted?
Cathal Garvey
people take it as a digestive aid. Alternatively, you can use "papain",
a very similar enzyme from papayas, sold for the same reasons.
Alternatively again, look for "meat tenderiser", sold by some butchers
or supermarkets; it's usually just bromelain powder. :)
Mega
>MgSO4 to chilled exponential-phase cells, and add DNA, and leave on ice
>for 30 minutes, something will probably happen. But again, it *has* to
>be liquid broth for this to work well.
E.Coli is on LB-Agar @ 33°C for 5 hours.
Then I take one colony.
Dissolve it in cold 1/2 ml liquid LB (+PEG+MgSO4)
Add plasmids.
Chill it.
And ready.
I'm happy with one transformant per ug DNA .... ;)
Cathal Garvey
I don't know if it will work well from agar. My suggestion is; it's your
first transformation. Try to reduce any points where it *might* fail, so
that if it does go wrong, you'll find it easier to fix.
Nathan McCorkle
before I want to do the transformation, I take 1mL of the overnight
culture and add it to 19mL fresh media. Shake at 37 for 2-3 hours,
then use that solution to do CaCl2 or PED/MgSO4 protocols.
Here is a CaCl2 guide that is well written by a professor of mine.
I've done this, it gives insane numbers of colonies, it DOES work when
you follow the steps. You will save yourself a lot of time by
understand WHY you're doing each step of a protocol, and what the
implications of adjusting parameters may be... or you'll never do
anything new... the difference between a car mechanic and a car
parts-changer is that the mechanic understands the machine and can
thus diagnose new problems and/or surmount unforeseen challenges.
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/21Transformation.pdf
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Mega
>PEG-3350. I have almost a kilogram of laxatives because I wanted to make
>the most of the horribly priced shipping. :)
That would be really really great... How much would I need to do a
transformation?
I think this ammount should well fit into an envelope... Sending an
envelope should not be too expensive, I think?? (Although I don't have
experience sending letters off-country)
By the way, could you help me out with some (very little, just for one
transformation) LB portion??
What would be an adequate recompense? About 7-10 € and mailing costs?
Mega
LB medium. I came home with 10g powder for making 1L. ;)
As I arrived home, I saw: She wrote "LB - Agar".
So it's probably not pure LB, but there's also agar in.
Does that matter when I feed the bacteria after transformation -
before adding Ampicillin?
Cathal Garvey
Then sterilise by pressure cooking for 20 mins.
Mega <masters...@gmail.com> wrote:
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Mega
Mega
I usually store bacteria in the basement. At the moment that should be
around 5-7°C cold....
When I take them out of the basement and put them in 33°C water, how
long will they need to grow exponentially? I think when exposed to
such cool conditions they surely will minimize growth.
Maybe it would be better to take them out of the basement 3 days
before handling them (e.g. transformation), so they get used to warmer
conditions. And then put them in 33°C (better 37, of course) warm
water for 3-5 hours (as usual)?
(Maybe this was one of the errors I did when transforming?)
Cathal Garvey
However, they will take a little time to "wake up", the so-called "lag phase". The lag phase will be longer for cells just out of the fridge than for cells grown from a just-exhausted overnight, so you'll have to wait longer before they are ready.
For ease of guessing, assume your cells are ready when the broth is cloudy when swirled, but not opaque! If it looks like cloudy lemonade, still transparent but cloudy, it's probably about right.
Mega <masters...@gmail.com> wrote:
Mega
again, and after some hours I opened it and the whole petri dish was
full of water. That may pose a problem to the e.colis to grow.
Now I think I have to get my coffe cup incubator tested in its future
environment. I.e. with petri dish inside.
Dakota Hamill
Mega
The fridge cooles until 7°C, then waits until temp reaches 8°C .... So
the bacteria get warm... cold... warm ....cold (cold in this case
means not so warm)
Dakota Hamill
Mega
fridge does not cool - temperature behind is on the decline. Then it
cools again ....
Dakota Hamill
Ah I see, you are right. It won't be a steady stream of warm air.
Brian Degger
You can put an aquarium heater in a large jar with water. Without water the heater will overheat and break the glass as the thermostat still is sensing cool air.
Cheers Brian
On 25 Feb 2012 18:19, "Dakota Hamill" <dko...@gmail.com> wrote:
Ah I see, you are right. It won't be a steady stream of warm air.I thought people had success using aquarium heaters without water as well?
--
Mega
itself.
that's an idea. A big container of air, therein a sammer container
with water and the heater.
Nathan McCorkle
if you incubate right-side-up the top might be cooler than the bottom,
and water vapor could condense, then droplets could fall on your agar
surface, splashing bacteria around and/or dilute things.... not good!
so always incubate upside-down!
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Paul Sian
Been lurking here for a while soaking up the information.
I have a question on what microscope to get to start off with. I read about the 1000x power being good for seeing better details of cells is that something I should consider or would something will lesser power work? Also I want to get my two kids (6 and 8 years old) involved with this as well as the have expressed an interest.
So what is a good microscope to consider and what are some good sources? I don't want to go crazy overboard on the microscope but don't mind spending a little extra for quality.
Thanks
Paul Sian
Avery louie
Check out american science and surplus. You want 400x at least for bacteria, at 400x they look like tiny dots swimmong around. 1000x is nice, just tenderness you need immersion oil. Oil is cheap, just don't forget to get it. On a macro level, you can look at interesting colony morphology pretty easily with a dissecting microscope.
--A
Nathan McCorkle
much, the last 1000X 'scope I got, I paid U.S.D $40 via craigslist.
There are smartphone apps that let you setup searches, and the app
polls every few hours and alerts you when new items come up. You might
have to wait a few weeks/months, but I think its one of the best ways
to get a good deal.
You can easily test a microscope's quality by placing your cell phone
under the lens with the backlight on, the three colored pixels should
be clear and not wavy/grainy (indicating bad lenses or dust in the
optical path).
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Patrik
website. I'm sure he'll be along shortly ;-)
http://micro.sci-toys.com/select
Johannes Debler
>
> http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/21Transformation.pdf
>
404 Error - Page not found
Unfortunately it doesn't seem to be available anymore. If you have it,
could you please upload it somewhere else?
cheers
Nathan McCorkle
http://people.rit.edu/rhrsbi/GEPages/LabManual.html
Google "rit rothman" and this is on his genetic engineering page
Simon Quellen Field
Cory Tobin
higher magnification. High mag doesn't get you any extra information,
it just makes the image appear larger. If you have high magnification
and low NA you'll just end up with a really large, blurry image.
In addition to Simon's advice that scopes without condensers are toys,
I would add that if the scope comes with an objective and they don't
specify* the NA of the objective, just the magnification, it's
_probably_ a toy. If they don't tell you the NA, it's probably
ridiculously low. The manufacturers of toy microscopes like to
advertise stupidly large magnifications, which doesn't tell you
anything about how small it will resolve.
* Sometimes the NA is specified in parentheses next to the
magnification. 60x (0.9) would mean 60 times magnification with a
numerical aperture of 0.9.
-cory
Simon Quellen Field
Mega
transformation. And guess what.
Monday I'm doing it.
But there are still a few questions...
When I make an LB Amp Agar Dish, I first mix water and LB Agar. Then I
heat it up. I think making 50 ml will be enough for one or two petri
dish.
Then I have to sterilize it. Will 3 mins in the microwave do the job,
or will I need to put it into the autoclaveur for 20mins as
recommended?
And, at <55°C I have to add the ampicillin which is not heat
ressistant.