PCR Cloning - primer design and CpG methylation (Jun/30/2004 )
I had to insert a 1.5Kb piece into 2 different expression vectors pEGFP-N1 and pHM6. I PCR'd the insert from a clone I bought from ATCC with the primers for the restriction sites for the 2 vectors (EcoR I and BamH1 for pEGFP and Eco RI and Not 1 for pHM6).
These primers also had the the kozak sequence on their forward primers after the restriction sites. The 5' end of the primers had an 'ATT' sequence added. So, it will be ATT-restriction site-kozak-primer in that order for the forward primers and ATT-restriction site-primer for the reverse primers.
I have been getting the correct size bands in each step and also the colonies, but there is no insert in them. Please advise me and let me know if these primers are OK. Also, I heard about blocking of cleavage by CpG methylation in Not 1 enzyme. Has anyone encountered this kind of blockage and what is the solution?
I have also tried TOPO TA cloning with the same primers without the ATT and the restriction sites, but have not got any results!
It sounds to me like your primers are not the problem if you say you are getting the right pcr product. However, if you are digesting your pcr product for ligation, the emzymes may not cut- sounds like you only have 3 bases (ATT) at the end, this is too short, enzymes (with some exeptions) require at least 6 bases to from recognition site to cut properly.
Also, the reason you are getting colonies could be
1) Your plasmid is self ligating- this is only possible if your 2 enzymes generate compatible ends, check this eg. at New England Biolabs site
2) You have uncut plasmid in the ligation mix- do you purify your cut plasmid before ligation?
About methylation blocking restriction: again, check NEB site, it will say if Not I is affected. Is your DNA methylated? Normally DNA propagated in commonly used bug strains is not methylated- check your strain.
Thank you kant0008!
Apart from the ATT sequences which you feel may not be enough for cutting, is the primer OK (provided all the other parameters, GC, Tm, etc. are fine)? Is the Kozak sequence needed or can I just use the ATG in the sequence?
The NEB site does say that Not 1 cleavage is blocked by CpG methylation. I always purify the digested vectors and inserts prior to ligation....but since I use Eco R1 and Not 1 for the digestion I havent actually 'seen' whether the Not 1 is cutting...coz what I am seeing could be the Eco R1 cutting!
The strains that I use for transformations are DH5 alpha and TOP 10 cells...I am trying to find what kind of strains I should be using if I have to use Not 1....
Thank you very much!
If you got a problem in designing primers , you can use Xpression Primer software . It will help you in designing tagged primers for high throughput cloning like Gateway , TOPO , BD in- fusion . You can also add restriction sites and other expression sequence of your choice .
I'm about to tear my hair out over a similar cloning problem.....Generated PCR product (1.5 kb) with KpnI and HindIII sites, but haven't been getting any inserts, with only 3 bases at the end of the site. My gut feeling is that the restriction of the PCR product hasn't worked, though i can't seem to confirm that this is the problem. I've been told that cloning large genes into plasmids is generally difficult...I would truly appreciate any suggestions that would give me at least 1 positive insert...just 1 will do....Please help...thanks.