Genetic Engineering Lab Manual
Nathan McCorkle
good, especially the first 30 or so pages of technique and background
info:
http://people.rit.edu/rhrsbi/GEPages/LabManual.html
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Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics
C.R.S.
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Albert Thompson
Good S@@t
Thank You,
Albert Thompson
School of EMTE, GWC Room 542
Tempe, AZ USA 85287-6106 Ph:480-965-0772
Em: THIN...@ASU.EDU (DSO: DARPA's DARPA)
-Albert Thompson III
"Scientific revolutions may be sensed in advance by scholars
with an exceptional intuition, but the site where such revolutions
take place is frequently a complete surprise."
..Dr. Esra Galun, RNA Silencing, 2005
Avoid fava beans. -- Pythagoras.
Cathal Garvey
I think you're referring to sodium borate as a buffer for electrophoresis. If memory serves, you want around 19.17g/L of borax.
Expect ladders and PCRs to run a bit quickly unless you dialyse them or leave them in the well to let the salt diffuse. It's about 50 times cheaper than tbe or tae though, and it interferes less with downstream reactions with purified DNA etc.
On 9 Mar 2011 17:17, "C.R.S." <edwin...@gmail.com> wrote:
Wow!, Thank you very much. This saves me a lot of time. I had planned to go to the Medical library here in Houston to look up methodologies. I remember reading somewhere about a much simpler borate buffer without tris that was cheaper and works better. Ed in Houston
On Wed, Mar 9, 2011 at 9:57 AM, Nathan McCorkle <nmz...@gmail.com> wrote:
>
> Here is the lab manu...
Cory Tobin
> If memory serves, you want around 19.17g/L of borax.
>
> Expect ladders and PCRs to run a bit quickly unless you dialyse them or
> leave them in the well to let the salt diffuse. It's about 50 times cheaper
> than tbe or tae though, and it interferes less with downstream reactions
> with purified DNA etc.
Borate inhibits ligase, so if you're planning on extracting a band and
ligating it, use TAE instead of SB (sodium borate) or TBE.
SB works well for bands under 3kb. Over 3kb the bands are very
diffuse and blurred together.
Under 3kb SB is great - you can run the gel in ~15 minutes and the gel
stays very cool.
-Cory
Cathal Garvey
Awesome useful man, thanks! I'll know to avoid it now, I was gonna use it to prep my new plasmid and would have wasted a lot of DNA trying..
On 9 Mar 2011 18:18, "Cory Tobin" <cory....@gmail.com> wrote:
> I think you're referring to sodium borate as a buffer for electrophoresis.
> If memory serves, you...
Borate inhibits ligase, so if you're planning on extracting a band and
ligating it, use TAE instead of SB (sodium borate) or TBE.
SB works well for bands under 3kb. Over 3kb the bands are very
diffuse and blurred together.
Under 3kb SB is great - you can run the gel in ~15 minutes and the gel
stays very cool.
-Cory
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balint
If you would like to get videos about basic lab techniques please have
a look to our blog:
http://labtutorials.org/
Moreover you are welcome to publish your protocolls or videos at
Labtutorials.org!
There are two ways to publish your protocoll:
1. ask to publish- this is straightforward, without any review.
2. ask for review- in this case you can suggest one reviewer and we
assign a second one. From this point on it is basically similar to the
process ofd scientific publication.
Cheers,
Balint.
General Oya
these guys have alot of user made video protocols as well.
Ryan
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Anselm Levskaya
restriction/ligase cloning anymore, but I don't seem to recall it
being all that bad in my reactions. I think it's a bigger issue if
you're purifying the DNA straight out of a low-temperature melting
agarose gel. If you're using a column-based method I think you get
rid of the vast majority of borate. In any case it'd be an easy
side-by-side test.
The nice thing about SB is that it's dirt cheap for DIY bio compared
to tris/edta buffers.
Re: band compression:
I noticed this reference that suggested making the SB buffer up at pH
6.5 in order to prevent high-MW compression. Making the SB buffer at
pH 6.5 gives me nicely resolved bands all the way up to about 10kb
and I'm now using it all the time (and running at 250V+ typically
takes 10min to get good resolution).
The Lithium borate buffer version would probably be even better, but I
haven't gotten any LiOH to try it out yet.
-Anselm
Cory Tobin
> restriction/ligase cloning anymore, but I don't seem to recall it
> being all that bad in my reactions. I think it's a bigger issue if
> you're purifying the DNA straight out of a low-temperature melting
> agarose gel. If you're using a column-based method I think you get
> rid of the vast majority of borate. In any case it'd be an easy
> side-by-side test.
I usually use the Qiagen gel extraction kit. I did a side by side
with TBE vs TAE, extracted a 2.5kb fragment from the gel, ligated
(cohesive ends) into a 3.5kb plasmid and transformed into Invitrogen
Top10 cells, 3 replicates. TBE gave 32% +/- 8% fewer colonies. With
cohesive ends this isn't much of a problem because you'll have plenty
of colonies. With blunt ends a 30% decrease could be a problem just
because there are very few colonies to begin with. But I found that
during the column purification if I do 2 washes with the QG buffer and
2 washes with PE, the problems goes away.
> Re: band compression:
> I noticed this reference that suggested making the SB buffer up at pH
> 6.5 in order to prevent high-MW compression. Making the SB buffer at
> pH 6.5 gives me nicely resolved bands all the way up to about 10kb
> and I'm now using it all the time (and running at 250V+ typically
> takes 10min to get good resolution).
Great news! I'll try that out this afternoon.
-Cory
Patrik
>
> The Lithium borate buffer version would probably be even better, but I
> haven't gotten any LiOH to try it out yet.
battery:
http://realitypod.com/2011/03/extracting-lithium-from-a-battery-cell/
Like Sodium, Lithium will react spontaneously with water to form
hydroxide and hydrogen gas, so be very careful when doing this!
Cathal Garvey
Great advice, thanks Anselm! Any idea how to deal with differences in salt between minipreps, PCRs, ladders?
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Nathan McCorkle
> Great advice, thanks Anselm! Any idea how to deal with differences in salt
> between minipreps, PCRs, ladders?
>
ethanol precipitation? the lab manual I posted says to adjust the
volume to 0.1mM NaCl, then add two volumes of ice cold EtOH, ice for
15 mins, then centrifuge for 20 mins and decant, dry, resuspend in
buffer
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Nathan McCorkle
consistently wrong units (microliters being represented with ml)... my
Prof says this happened when he changed fonts... I might be able to
get an older version to post, then comparing the two versions should
clear things up.
I hope this message propagates before people are confused with strange
amounts and concentrations.
-Nathan