linear tandem repeat using BstXI problem - (Dec/01/2006 )
Hello Molecular biologists,
I am trying to generate a tandem repeats of a 100 bp construct such that each repeat is in the same orientation.
I make the 100 basepair construct with BstXI sites at the two ends.
I am trying to digest this with BstXI and ligate such that I get linear tandem repeats in the same orientation.
However when I do the experiment I am only getting a dimer.
I digest with BstXI for 2 hrs at 55C. Column purify. Ligate at room temp. for 2hrs.
Any help is welcome.
thank you
Very high concentrations of inserts will favor concatamers. I would probably ligate the insert only at high concentration, then do a second ligation at low concentration with a mixture of the vector and the result of the first ligation. If you heat kill the ligase and run the ligation results on a gel you can evaluate the success of the strategy. Don't do this if you are using a PEG containing (quick ligase) buffer, but there is likely no reason for you to be using one in any case.
I presume you have designed the cut sites for BstXI such that compatible ends are left after the digestion. If not, then that is your problem, and changing ligation conditions won't fix it.
Thanks.
There is no "vector" in my experiment.
I have a linear 100bp linear PCR fragment with BstXI sites at the two ends (with ~4 bases flanking the BstXI) site.
I digest this 100bp fragment with BstXI.
Column purify.
Ligate at room temperature for 2hrs.
Only a dimeric species observed.
Also, why does one need to heat inactivate the ligase to check the ligation results on a gel?? I don't quite follow this.
Thanks again.
I presume you have designed the cut sites for BstXI such that compatible ends are left after the digestion. If not, then that is your problem, and changing ligation conditions won't fix it.
There is no "vector" in my experiment.
I have a linear 100bp linear PCR fragment with BstXI sites at the two ends (with ~4 bases flanking the BstXI) site.
I digest this 100bp fragment with BstXI.
Column purify.
Ligate at room temperature for 2hrs.
Only a dimeric species observed.
Also, why does one need to heat inactivate the ligase to check the ligation results on a gel?? I don't quite follow this.
Thanks again.
It has been reported (i believe by phage434) that active ligase causes DNA to run slowly, possibly due to the ligase being bound to the DNA strand. I personally have not seen this effect. It could be dependent on the amount of ligase being used.
If you would check NEB technical pages http://www.neb.com/nebecomm/tech_reference...nucleotides.asp
you would discover that 2hr digest with yeild at best a 25% cut rate efficiency. I am not sure how many units of enzyme you are using, but the situation does not look good.
Well, you can try this yourself -- it's a trivial experiment. We find it makes a large difference. What are the precise cut site sequences in your primers (including overhangs)?
Another possible problem is inadequate cleanup of the PCR reaction prior to digestion. If there is active PCR enzyme (even in the absence of dNTPs) then the ends of the cut DNA can be chewed back. If there is dNTP available, even in small amounts, then fill in of your cut site can occur. The extreme version of cleanup uses proteinase-K to digest the PCR enzymes prior to DNA cleanup.