Troubleshooting transformation and ligation efficiency
Jeswin
still trying to figure out how to get this right and improve my
technique. This cloning stuff feels more like an art now.
Is it possible that the ligation was not so efficient? I'm not having
the most confidience in my ligation calculations. How do I check my
ligation to see if they worked? Run a gel?
From the 1 or 2 colonies I got, the inserts were worng orientation.
The plan is just to have them sequenced to make sure the PCR
mutagenesis worked. If so, I will just cut and re-ligate again. Right
now, it takes too long to go from PCR => colony PCR-screen =>
orientation screening. As long as we know the insert is correct, it is
not so hard to get it into correct orientation later.
Cathal Garvey
ligation floating around involving overnight 4C, when in fact it should
only take 30 minutes at RT. Buffer *must* be ok though; it can go bad!
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Jeswin
> How did you do the ligation? There are lots of mythical protocols for
> ligation floating around involving overnight 4C, when in fact it should
> only take 30 minutes at RT. Buffer *must* be ok though; it can go bad!
>
Made the mix. Ran it on the PCR machine for 15 minutes at 25 degrees.
Will have to check the buffer expiry and stuff.
Nathan McCorkle
they probably continued cutting during and after ligation.
Incubation time for ligations, from what I've heard, depends on the
buffer used. When I've done ligations its been in the fridge using the
buffer recipe (at the end) and procedure in this manual:
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/20%20Ligation.pdf
I was taught to do multiple controls at ligation time though, and
check with a gel immediately after to diagnose any problems before
moving on to transformation. Without controls, you may lose valuable
data/info for diagnosing problems later!
Here's a question I posted regarding 'quick' vs normal ligations:
http://groups.google.com/group/diybio/browse_thread/thread/b171ff2db4d87e6f?pli=1
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
Jeswin
> Did you deactivate your restriction enzymes before ligation? If not
> they probably continued cutting during and after ligation.
>
a gel and purified the DNA.
> Incubation time for ligations, from what I've heard, depends on the
> buffer used. When I've done ligations its been in the fridge using the
> buffer recipe (at the end) and procedure in this manual:
> http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/20%20Ligation.pdf
>
Our buffer was frozen and I just thaw it. Does the thaw cycle
negatively affect the buffer?
> I was taught to do multiple controls at ligation time though, and
> check with a gel immediately after to diagnose any problems before
> moving on to transformation. Without controls, you may lose valuable
> data/info for diagnosing problems later!
>
What kind of controls do you use? I have a negative control using just
the vector and no insert. However, I don't run any gels of these after
ligation. I do plate all reactions though.
Nathan McCorkle
> On Fri, Feb 10, 2012 at 1:41 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
>> Did you deactivate your restriction enzymes before ligation? If not
>> they probably continued cutting during and after ligation.
>>
> What do you mean by deactivate? After cutting, I ran the reaction thru
> a gel and purified the DNA.
Deactivating would be 65 C bath for a while, unless the enzymes were
thermo-tolerant, then you'd need a phenol:chloroform extraction.
Gel purification works too!
>
>> Incubation time for ligations, from what I've heard, depends on the
>> buffer used. When I've done ligations its been in the fridge using the
>> buffer recipe (at the end) and procedure in this manual:
>> http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/20%20Ligation.pdf
>>
> Our buffer was frozen and I just thaw it. Does the thaw cycle
> negatively affect the buffer?
Did the portion you use completely thaw completely so you know it was
mixed evenly (or did you have say 500ml frozen, let it thaw just
enough to pull off 1ml)?
>
>> I was taught to do multiple controls at ligation time though, and
>> check with a gel immediately after to diagnose any problems before
>> moving on to transformation. Without controls, you may lose valuable
>> data/info for diagnosing problems later!
>>
> What kind of controls do you use? I have a negative control using just
> the vector and no insert. However, I don't run any gels of these after
> ligation. I do plate all reactions though.
Just like the ones in the PDF I linked to, basically to make sure the
insert to vector ratios are correct, making sure self-ligation doesn't
happen (if you use BAP).
Did you plate dilutions of your transformant solutions, so you can
calculate transformation efficiency?
Jeswin
comparing vector and insert and found insert to be 2x intense as
vector. Vector size was 8075 and insert was 1317 bp. I want a 1:2
ratio, vector:insert. How much insert should I have used? I'll look my
answer, but I think it was about 2 uL vector and 1.8 uL insert. I
didn't do the nanogram calculation but used the method here:
http://www.methods.info/Methods/RNA_DNA/ligation_simple.html
Gel image is attached
Nathan McCorkle
well as using imageJ's intesity measure tool... i got that you have
about 8-10X insert to vector... assuming you resuspend in equal
volumes post-gel (you said you're gel purifying right?)
attached is my spreadsheet
Nathan McCorkle
transilluminator might not have even illumination, something a
spectrometer with a single pixel doesn't really suffer from. Do you
have a nanodrop or equivalent? The best thing to do would be
resuspend, then quantify with UV... as the solution tested is the
ready to use solution, whereas you might have losses in the gel
extraction protocol downstream of the gel imaging.
On Mon, Feb 13, 2012 at 11:54 AM, Jeswin <phill...@gmail.com> wrote:
helicase
was your PCR product fresh? Those 3' A overhangs tend to fall off with
time...
Cathal Garvey
You mentioned that you gel-extracted your DNA at least once before
transformation; extracting the insert before ligation, was it?
Were you using UV illumination when you gel-extracted it? Even transient
(less than ten seconds) UV illumination can cause tenfold reductions in
successful clones downstream, by cleaving your linear DNA and causing
thymine-dimers that may impair DNA establishment in transformed cells.
Try to manage without UV and see if it fixes the problem. Some suggestions:
A) Use a blue-light dye like Sybr-safe or nbsbio.co.uk's "SafeView" (it
*does* fluoresce under blue, but with less activity)
B) Find an enzyme that cleaves the fragments you don't want while
sparing your insert; that'll reduce spurious transformants if you forego
a gel entirely.
C) If the fragments you are gel-purifying away are spurious PCR results,
amplify a more unique, wider region around the target sequence first,
then purify that and PCR up the sequence you want using the original
primers once it's the only template in the mix. If it's the only visible
result on a gel sample, try cloning with just this sample.
If you forego gel purification, remember to heat-inactivate the enzymes
you're using; most should break after 20 minutes at 60C, but some may
need 80C. A rare few are thermostable enough that you'll have to do a
PCR-cleanup or alcohol extraction instead to get rid of the enzyme.
--
Pieter
ligation protocol. Some only ligate for 3 hours at 16C others over
night (at least 8 hours that is). We all use the same Fermentas
ligation buffer and T4 ligase.
I normally aim at a concentration of 100 ng/μL DNA and add about 5 μL
to the competent cells.
How do you transform/incubate your cells after adding the ligation
mix?
To check whether the ligation reaction was succesful, you could PCR
the result of your ligation reaction with primers designed for the new
insert.
What vector are you using, and what is the size of your insert
compared to the size of your vector?
> >> I want to check my last calculation for ligation. I had run the gel
> >> comparing vector and insert and found insert to be 2x intense as
> >> vector. Vector size was 8075 and insert was 1317 bp. I want a 1:2
> >> ratio, vector:insert. How much insert should I have used? I'll look my
> >> answer, but I think it was about 2 uL vector and 1.8 uL insert. I
> >> didn't do the nanogram calculation but used the method here:
> >>http://www.methods.info/Methods/RNA_DNA/ligation_simple.html
>
> >> Gel image is attached
>
> >> On Sat, Feb 11, 2012 at 3:52 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
> >>> --
> >>> Nathan McCorkle
> >>> Rochester Institute of Technology
> >>> College of Science, Biotechnology/Bioinformatics
>
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Jeswin
purified my ligation with a gel. Actually, I don't. I had gone
straight to the transformation. Today, after the ligation, I
deactivated for 5-10 minutes at 60 degrees. Maybe it will help. I also
noticed that I was only plating 100 uL of the tranformed cells. Today,
I am going to plate all 300 uL of it. Also, after plating, I did not
let the plates rest for 30 minutes before 37C incubation. I am guess
that cells were not able to enter the agar?
With these 3 changes, maybe I will have better luck.
Nathan McCorkle
> I just noticed some things that I did not do. Previously, I said I
> purified my ligation with a gel. Actually, I don't. I had gone
> straight to the transformation. Today, after the ligation, I
> deactivated for 5-10 minutes at 60 degrees. Maybe it will help. I also
generally i've heard 30 minutes for deactivation of nucleases, I've
gone from ligation straight to transformation with success before
> noticed that I was only plating 100 uL of the tranformed cells. Today,
> I am going to plate all 300 uL of it. Also, after plating, I did not
generally you would have 1-2mL of cells if you add LB or SOC media
after the heat shock, then incubate in a shaker for 45-60 mins at 37
C... then make some serial dilutions of this transformant solution,
and plate on multiple plates... the dilutions allow you to accurately
do colony counts as there will likely be plates with innumerable
colonies crowding each other.
> let the plates rest for 30 minutes before 37C incubation. I am guess
> that cells were not able to enter the agar?
I've never looked into or heard about E.coli 'entering' agar, I don't
think they posses agarase.
>
> With these 3 changes, maybe I will have better luck.
Good luck!
Cathal Garvey
heat-killing the ligase before transformation. Like many DNA-affecting
enzymes, it "rests" on DNA when inactive, and your DNA can get bogged
down by excess ligase, apparently.
Or at least that was the theory; all they could say for certain is that
it reduced transformation efficiency. It's just as possible that ligase
goes into the cell with the DNA but then occludes the origin of
replication, causing plasmid loss. Something odd, anyway.
Anselm Levskaya
I -highly- recommend using a ligation buffer that contains a
high-molecular-weight PEG additive to induce "molecular crowding". A
T4 ligation reaction w. PEG will run to completion within 15min-30min
and beat a non-PEG ligation by an order of magnitude or more (based on
the resultant number of colonies / uL of ligation mix). These are
sometimes marketed as "quick ligation" kits, but the only substantive
difference is the presence of PEG.
I've never found removing the ligase to be necessary to obtain high
efficiency transformations.
The only time you need to purify anything is if you're going to do an
electroporation, in which case you have to get rid of the salt. (this
can also be done by little dialysis pads vs. full purification)
-A
Nathan McCorkle
completely inhibiting transformation:
http://groups.google.com/group/diybio/browse_thread/thread/b171ff2db4d87e6f?pli=1
Good Call Anselm on the "molecular crowding" idea, that makes pretty
good sense (googling "molecular crowding peg" shows a lot of results
on this.
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Cathal Garvey
Nathan McCorkle <nmz...@gmail.com> wrote:
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Jeswin
some toxic product from the insert. Also, I ran a gel with my
ligation, negative, vector, and insert (in that order). I am not sure
what to make of it. (Also, boss says you're not supposed to see
anything if you run a gel of the ligation, whatever that means?)
Nathan McCorkle
> So it all failed again. I don't know what could be the problem except
:(
> some toxic product from the insert. Also, I ran a gel with my
> ligation, negative, vector, and insert (in that order). I am not sure
> what to make of it. (Also, boss says you're not supposed to see
> anything if you run a gel of the ligation, whatever that means?)
Sounds like your boss is mistaken... my genetic engineering prof
specifically mentioned to run a 'baby gel' of the ligation reactions
to diagnose the outcome prior to transformation, so you can correlate
transformation outcome with DNA ligation... if you have ligation
product then your problem is the transformation... if you have no
ligation product that undermines transformation ever working
Jeswin
was sequenced correctly but inserted into vector in wrong orientation.
The plan is to cut and religate and get a colony in the right
direction. Well, in cutting, I added AP to both reactions.
Nathan McCorkle
Jeswin
> AP as in Alkaline Phosphatase?
>
had no colonies today. Still have no idea why the efficiency of the
other transformations are so low.
Jeswin
> was sequenced correctly but inserted into vector in wrong orientation.
> The plan is to cut and religate and get a colony in the right
> direction. Well, in cutting, I added AP to both reactions.
>
religated again since the original direction was wrong. So I made 2
rxns to cut and purify the vector and insert for ligation again. My
mistake was adding Alkaline phosphatase to both the vector I want and
the insert I want. I only needed AP in the cut plasmid to keep it from
religating. I didn't understand that previously.