Hello all!
I am currently trying to clone a 120bp fragment. However, I am getting a low yield. I am using 2ul of 50ng DNA for the PCR reaction. I have also tried a gradient and I am using the best temperature I found for this primers. I am also using DMSO and Phire Polymerase.
In addition, my primers are 22bp long plus 5bp of restriction enzymes, and three nucleotides to enhance restriction activity.
Should I just order new primers?, However, I don't have that much room to vary them as I am amplifying signal peptides, should I use this resulting reaction as a template as I am also getting some genomic DNA amplification?
THANKS!
Instead of struggling with PCR and enhancing the product, we make 50ul volume of PCR reactions (4-5 tubes) and load the whole (50ul) to the gel. Then we purify the desired product (120bp in your case) from gel by gel purification kit.
After digestion of the eluted product, we again purify the PCR product (digested) from reaction mixture itself by same purification kit and use for ligation.
This protocol always works for us.
However, I have a very low yield. If you look at the first gel, on the left side there is barely any product.
I'm assuming you've already tried increasing cycle number? You could always try purifying your PCR product and reamplifying it.
it may be that your dna has inhibitors in it ( or even contains rna so there is less dna than you thought) so you might try titrating dna in the pcr at your optimum conditions say from 1/5th to 3 times as much dna . Check also that the addition of a restriction site has not created conditions where a hairpin in the primer is possible making that primer less useful
Dear Margarita,
Please follow the suggestions by Paul and others. I assume that your templates do not contain inhibitors or are otherwise good for PCR. I dont know what template you are using. if you are using a circular supercoiled template, then try to linearize it with a single cutter RE that cuts your template outside the primer target region. Use this linearized primer for PCR. Even if you are not using a circular supercoiled template, you can digest it with an RE to reduce the complexity and enhance primer binding. Just make sure the RE sites are outside the primer target region.
Best of luck
SD
Dear Margarita:
You have already got a number of good recommendations, but for me the best one is to check for quality of the DNA template, just titrating the DNA as recommended by Datta, and may be also to linearize your template by some Restriction Enzyme, outside the region to be amplified. Be sure to inactivate the RE by heating at 100ºC for 5 min. before to use the DNA as template.
Good luck
Dear Margarita,
In order to enhance PCR reaction you should optimize not only the temperature you should also the time for each step in a PCR cycle.
clean up your template with ethanol precipitation and let the start concentration be 100 ng, plz also decrease you annealing temperature, to increase non specificity find which annealing temp work for maximum product generation. then carry out touchdown PCR from higher annealing temp to lower annealing temp (optimal temp)
Dear Margarita
It could be because of eight extra nucleotides added to primers for restriction enzyme sites. In such cases we set PCR starting with first 10 cycles at the annealing temp. calculated based on primers sequence without adding extra nucleotides. then increase the annealing temp by 1 degC each cycle till 65-68 degC depending upon the annealing temp. calculated based on entire primers sequence (including extra nucleotides added fro restriction site). Which typically follows for 15-20 cycles at highest annealing temp. set. Mostly it works. If you send the primers sequence and the cycle conditions you are following, may be I can help by suggesting the cycle conditions.
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