Create an artificial restriction site for a endonuclease and 53bp long primer? - (Dec/30/2008 )
Hi everyone!!! Im using a 53bp long primer, it creates an artificial restriction site for Bsh1236, its too long because the enzyme cut at 52bp reconigtion site and so it can be seen in an agarosa gel. I already have results but it worked for 10 of 90 DNA. The annealing temperature is 63ºC and its G/C content is 64%. So how it creates a restriction site??? and how is the primer working if the annealing temperature should be higher for a primer that long???? I used DMSO and some other agents to make it work, does long primer needs more DNA available that shorter primers?? Thanks
Could you please rephrase your question. I am afraid I don't quite understand.
Only the part of your primer that binds to the template is counted towards the tm calculation.
You may have a 100bp primer, but if only the last 18 bp actually bind to the template, then the tm that you would use is based only on the 18bp.
long template do not need more template. treat them exactly the same as you would a shorter template
the tm that you should aim for is around 58 C to 62C.
Only the part of your primer that binds to the template is counted towards the tm calculation.
You may have a 100bp primer, but if only the last 18 bp actually bind to the template, then the tm that you would use is based only on the 18bp.
long template do not need more template. treat them exactly the same as you would a shorter template
the tm that you should aim for is around 58 C to 62C.
Thanks for answering. Well there are 3 questions. This primer works, but I dont know if all the primer binds to the template, its sequence has 8 mismatches with the NCBI reported sequence. 1) So its not important the large of the primer?
2) "long template do not need more template". I worked with 3 set of primers, the other two (near 20bp) worked perfectly for 33%. Im using 2 DNA sources, according to some investigations: the total DNA from Saliva actually has 68% of human DNA while DNA from Citobrush has only 11%, the rest are from bacteria. So I tended to think that maybe more DNA could helped the PCR because all Saliva DNA turned positive, while all DNA from citobrush turned negative. So the difference between the long primer with the others is actually its 53bp and the PCR temperatures and time. ??
3) Im not able to sequence the DNA. So Im using RFLP. The problem is that there is not reconigtion site for any enzyme. So this primer creates an artificial restriction site that separates one alele from the other.
Mecanism: the enzyme`s recognition site is located at 52bp, so the last bases are the critical ones.
I dont know how it does it, but it works. Thanks for taking your time for answer!!!!!