Trouble with ligation Attn: Maxy Mariasegaram

[Harry Witchel]

Dear Dr. Mariasegaram --
	Your request for information was extremely crisp, clear, and to 
the point.  I agree with Gregg Nattrass in all the recommendations he 
made concerning your protocol, and I will make a few more 
recommendations.
	First, you asked if your digestion protocol for making a genomic 
DNA library from camel was appropriate, and I suspect that it would be 
more traditional to partially digest with MboI than to digest to 
completion.  Assuming that camels have about the same number of 
nucleotides as humans in their genome, if you digest to completion with 
MboI and end up with mostly fragments in the 1-2 kb and smaller range, 
you will need to screen a minimum of 1 million colonies (that assumes 
that every piece of DNA is expressed in exactly one colony).  In most 
library screens, some DNA species will be over-represented, while others 
will only occasionally appear.  Given your digestion to completion, it 
sounds like you will be screening about 5 million colonies, which is 
quite a lot of work.
	Two alternatives would be to use longer inserts by partial 
digestion with MboI.  These longer inserts can be up to 25-40 kb if you 
use cosmids or phage instead of plasmids.  Or, you can make a cDNA 
library from only one tissue, which will restrict which tissue you are 
looking at, but will reduce the number of possible clones you will have 
to screen through.  A final possibility (if you are trying to clone from 
a camel a gene which has already been isolated in another animal) is to 
use PCR.  If you can possibly solve your problem with PCR, you should 
definitely consider doing PCR before library construction and screening, 
even if it means traveling abroad to another laboratory to borrow a PCR 
machine.

	As for your ligation problem, you do not say whether you gel 
purified your 1-2 kb insert fragments, or how you eliminated the MboI 
activity from your camel DNA.  If the MboI was still active in your 
ligation mixtures, then the ligation  reaction would be rapidly undone 
by the MboI.  If you gel purified your camel DNA fragments, my first 
suspicion would be that your camel DNA is slightly contaminated with 
inhibitors of ligation from the gel itself.

To eliminate gel inhibitors from DNA: there are many many commercial 
kits available to do this (eg Qiagen, Promega, etc).  My personal 
favorite (usual disclaimers about no commercial connections) is 
(starting with <5 ug DNA in 50-100 ul of TE) to cool your DNA on ice 
(or in fridge) for at least 1 hour, then spin at 10000-15000 rpm for 10 
minutes, and move supernatant to a new tube (leaving behind any pure 
agarose pellet at the bottom).  Then heat your DNA to 65C for 25 minutes 
(to dissolve any remaining agarose), cool it to 40C and add 1/10 volumes 
of Beta agarase buffer and 2 ul of beta agarase (I buy this from New 
England Biolabs), and let the beta agarase digest it overnight at 40C.  
The next day phenol:Chloroform extract (some people prefer first to just 
phenol extract, then to phenol:chloroform extract, but I find it works 
with just one extraction).  Don't forget when doing phenol chloroform 
extractions on short (<10 kb) pieces of DNA, to really vortex vigorously 
-- I vortex for three burst of 10 seconds.  Finally you can ethanol 
precipitate your DNA (using NaOAc), wash it with 70% ethanol, and then 
resuspend in whatever volume is appropriate for your ligation.

	One final thought about your ligation: although ligations do 
work fine at room temperature for 5 hours, for high efficiency 
situations (such as making a library) it is a good idea to do your 
ligation overnight at 16C.  The room temperature ligations are more 
appropriate for moving a single purified species of DNA to another 
vector.
	All the best,
		Harry


Dr. Dr. habil U. Wernery wrote:
> 
> I am trying to set up a genomic library of camel DNA using SK+ vector.
> Unfortunately, I have had no luck in obtaining recombinant plasmids.
> 
> My approach involves restriction of SK+ with BamHI followed by ligation
> to 1-2 kb fragments of genomic camel DNA generated by complete digestion
> with MboI.
> 
> Is this a suitable approach? I had set up five ligation reactions as
> follows:
> 
> Tube #1:  dephosphorylated vector only  (negative control)
> 
> Tube #2:  vector that was linearized but not dephosphorylated (positive
> control)
> 
> Tube #3:  dephosphorylated vector + 1-2 kb fragments + 15% PEG
> (polyethylene glycol)
> 
> Tube #4:  dephosphorylated vector + 1-2 kb fragment
> 
> Tube #5:  dephosphorylated vector + 1-2 kb fragments + 25% PEG
> 
> Tube #6:  dephosphorylated vector + pUC 19 control plasmid (for
> transformation efficiency determination)
> 
> The tubes were made up to volume (15 microlitres) with water, ligase
> buffer, and  T4 DNA ligase. The mixtures were incubated for 5 hours at 20
> degrees Celsius and subsequently transformed into competent DH5 alpha
> cells (from Gibco, BRL). They were then plated on LB-Amp plates and
> incubated at 37 degrees Celsius  overnight.
> 
> Examination of the plates the next day revealed that transformation
> efficiency was good (tube 6).  As expected the tube with the
> dephosphorylated vector produced no colonies (tube 1). Tube 2 containing
> the linearized but not dephosphorylated vector control produced colonies.
> This would indicate that the dephosphorylation, ligation and
> transformation controls worked.
> 
> How can I account for the fact that no colonies were observed for
> ligation of vector  with the genomic DNA? I would be grateful if someone
> can recommend an alternative approach or suggest improvement in the above
> set up.
> 
> Thank you very much for your time and consideration. Please reply  by
> e-mail to (Attn: Maxy Mariasegaram):
> 
> microbio at emirates.net.ae
> 
> Sincerely,
> Maxy Mariasegaram