I have a problem on primers with restriction site. Here are my primers
Forward_XmaI: TCTATA(CCCGGG)GACC.... (34 mer, GC=52.9%)
Reverse_SalI: AACACT(GTCGAC)TTACA...(32 mer, GC=43.8%)
The template is a recombinant plasmid with my gene of interest (~5kb). I successfully got the 5Kb product with using Phusion Polymerase with HF buffer and 3% DMSO. The cycle conditions were 98C 30s (initial denaturation), 98C 10s, 60C 25s, 72C 6min (35 cycles) and 72C 10min (final extension).
Then I inserted it in pcr8/gw/topo TA vector (I added "A“ following the manufacturer's instruction), and it worked find. However, I could only cut it with SalI, but not XmaI. I thought maybe it was the problem of XmaI, but I used it to cut my target vector (yeast vector pGAD-C1), it worked perfect. I also used SmaI (share site with XbaI) on TA vector, no luck still. So I am pretty sure, it was the problem of the restriction site, maybe because the secondary structure covered the restriction site or something. So could anyone help me with it? Thank you so much!!
you are probably right in your assessment but Xma1 is very sensitive to salt concentration so template needs desalting ,preferably cutsmart buffer if you are using NEB enzyme and cut overnight
XmaI and SmaI cut the exactly same sequence at a different location (3' overhang and blun end respectively). Maybe, as Paul said, you have to much salt, do some PCR purification. Did you sequence your PCR, at least to verify the primers sequences (you can do a TA cloning or some equivalent / pGEM / pJET ...).
SmaI create a blunt end and the enzyme is active at 25C or 37C, NEB and Life technologies respectively (so be careful), I prefere to use XmaI that create a 3' overhang and is active at 37C. Also verify you used the correct buffer.
Once I had a problem with my primer itself, one base was incorrect, but it was inside the recognition sequence of my restriction enzyme. I order a new batch of primer and it work perfectly. And at the same time, I had a mutation inside the target plasmid and it was impossible to cut it, only ont restriction site was impact. Both happened to me at the same time.
Have a Good Luck
Hi, the first thing you would do is to sequence using a primer that anneals inside the insert going outside(to the vector backbone).There might have been a mistake during synthesis.
Second get a restriction map of the entire primer(some enzyme do not work when placed in the proximity of other sequences)
Lastly try also to optimize your salt concentration though i doubt if this can result in non digestion at all.
I would agree with Steven. If salt concentration is an issue, you would still see partial enzyme activity.
Do the following:
1. Double-check the sequence of your primer
2. Perform colony PCR on a bunch of colonies to make sure that they contain right-sized insert
3. If the answer to 2 is yes, sequence those colonies to make sure that the XmaI restriction site is intact
My guess is that something went wrong with the restriction site sequence, probably during primer synthesis.
good luck,
Hi, has your problem solved?
I think you should do DNA sequence with universal primer first to check whether there has mutation in restriction site, and then check the buffer you used.
Good luck with that
Hello,
You can try digestion with AvaI (C^YCGRG) to check restriction site. More quick than senquencing. AvaI cut 3 times in the original vector at position 561,1174,2078
So you must have 4 fragments with you insert.
Good luck
since you have good product it might be quicker to design a new primer with a new restriction site and reamplify your product and insert the new product into your vector
As suggested by the others if the change in salt concentration does not work it is better to redesign one of the primers which has secondary structures
- Badges
- Science method