Hi everyone .. I have great difficulty in cloning a promoter of a stem cell marker in an expression vector. the region is high GC percentage and in fact in the pcr, I had to use both adequate buffers and dmso.For cloning i needed to add some restriction tales on the primers. i changed various ligase protocols, and although pcr colony were sometimes promising, the control digestions and sequencing did not match .. i'm trying them all ... suggestions?
Hi Matteo, Is there a problem with the PCR or the Ligation ? From what I can tell you are using GC enhancement PCR buffer and additives like Betaine and DMSO to get your amplicon. You will need to add extra polymerase as the additives can reduce their efiiciency. If you are having trouble validating whether this amplicon is your GOI, you can tell by the size or if you want to be extra sure, you can digest it with a unique single cut restriction enzyme to see if the digestion remnants match the expected size if you were to digest your GOI alone.
Not Getting positive colonies after ligation could be due to
1. The digestion and purification of vector and insert were not efficient. (increase digestion time and use fresh buffers where the enzymes show their highest activity. Always gel cut purify the DNA to avoid undigested DNA contamination. Recheck if your insert primers have sufficient bases at the 5' end to allow for the enzyme to ocupy during digestion)
2. Molar ratio between the vector and insert was off. (try to keep the molar ratio around 1:5 or 1:7. Using higher ratios as well as lower ones retard efficient ligation)
3. Ligation buffer issues. (Ligation buffer needs to be fresh and free of preciptates. You can supplement 1mM ATP to the reaction if the buffer is old)
4. Phosphatase treat your vector to dephosphorylate the sticky ends and avoid self ligations. Sometimes false positive clones are unavoidable. Therefore, always plate an Empty vector + Ligase - insert control to know how many false positives are present in the reaction. If the number of colonies in the Vector + Insert plate are the same or slightly more as the Vector only plate; do not screen it as the chances of getting a clone will be slim there. The difference between the plates is usually stark if everything went according to the plan. If it is not so, change the ligation conditions and try again.
Are you getting amplification of a single, clean, correct-looking size band on a gel after you run PCR?
yes i do a gel clean up..first I have to do a digestion of the restriction tale on the primer
I keep it overnight...my doubt is that maybe the enzyme doesn't work so well and I can't see if there is still an uncut on gel.
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