I'm doing a site-directed mutagenesis on a pBABE puro with the HA-BRCA1 gene. I'm currently using the Superfi Master Mix kit with the following conditions.
50 µl reaction:
25 µl Superfi Master Mix
2.5 µl Primer Fw
2.5 µl Primer Rv
1 µl Template (25 ng/µl)
Conditions:
95°C 2 min
95°C 10 sec
50°C 10 sec
68°C 6 min
68°C 5 min
second, third and fourth step x 35 cycles
the plasmid is 10 400 bp. I ran the gel after DpnI digestion and I got a thin band in the loading wells, I thought it might have worked and I proceeded with the transformation but I got no colonies in the plates where I performed the digestion while in the control one without DpnI digestion I had colonies from the template. So there is something wrong with my PCR but I can't find any protocol online, I just followed the one from the kit. Has anyone ever used this kit? Please help me!
Did you try to perform phosphorylation and subsequently ligation of your PCR amplicon before transformation after dpnI digestion? Your amplicon doesn't contain 5' phosphate if you haven't phoshprylated your primers before performing pcr, for this reason you cannot circularize the template of your interest. I hope this is helpful for you.
Best wishes
Thank you, no i haven't, I followed the protocol that was performed in my lab and they didn't use the phosphorylation and ligation. i found kind of strange having lot of colonies in my control with polymerase, without dpnI and zero colonies in the control without polymerase and without dpnI. If there is the template there should be colonies in both of them right?
The classical procedure to follow to perform plasmid specific mutagenesis is:
1. Perform PCR with divergent primers to insert different nucleotides or deletion from the initial plasmid template
2. Gel extraction of the PCR amplicon to specifically purified the template of interest only
3. Digestion of the pcr purification with dpnI to digest only the template of origins, which contains methylation on DNA
4. DNA purification and phosphorylation of the pcr treated with dpnI
5. Re-circularization of the pcr amplicon by ligation.
Your control is the transformation of your PCR after dpnI digestion to evaluate the presence of contaminant plasmid of origin.
This protocol works very well in my lab!
Best wishes
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