Has anyone had previous success in amplifying Lactadherin from the addgene construct below?
https://www.addgene.org/46830/
I'm looking at the sequence and it seems that the starting sequence of Lactadherin has too large GC content (83% GC) and annealing temperature (TM= 82'C) for PCR. Please see attached image for the forward sequence.
We perform PCR using Q5 HiFi DNA Polymerase and compute the Annealing temperature based on NEB's online TM calculator. The maximum TM recommended is 72'C.
Could anyone recommend primers that had good success? Thanks.
Here is what you can try:
1-You never mentioned the tm of the rev primer but looking at the F primer seq
I would suggest to try run a 2 step PCR program at 72 preferably at 70 just to test if you get any amplification (increase the initial denaturation time).
2-PCR with additives (DMSO, or high GC enhancer from NEB) and run at slightly lower tm depeding on DMSO concentration you are adding maybe 67 would do depending on rev primer tm.
3-In case Q5 fails I have found phusion to perform robustly in difficult amplifications.
4-I case all else fails you may want to re-synthesize the fragment and lower the GC content to something more manageable (it is fairly cheap for short length frags).
Hi Hanna Alalam. Thanks for your recommendations. The reverse primer seems to be okay (see attached image). It's just the forward primer that's a little troublesome. I will try doing 2 step PCR and update with the outcome.
Please see the links attached. The primers are carefully listed hereArticle Identification of major milk fat globule membrane proteins f...
Article Milk fat globule-EGF factor 8/lactadherin plays a crucial ro...
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