Need to confirm PCR primers are bad - (Feb/26/2007 )
Hello everyone! I am having some problems with an RT-PCR reaction as I am only getting a small approxiamately 100 bp amplicon. It is also not a very tight band with some smearing. My target is 1.2 kb. I am guessing that my primers are bad and mispriming. They are supposed to be lyophilized, but in some of the unopened vials there is a small amount of liquid. They were purchased about 2 years ago, but I am in Peru and this all that I have to work with for now. I have run lots of PCR in the past and this is the first time I have had major primer problems. I need to show to the vendor that they are degraded. I have heard that I can run them on a polyacrylamide gel and if they are degraded they will show a ladder separating out the degraded primers. The two primers for my RT-PCR reaction are 22 bases and 27 bases long. Has anyone tried this? What concentration polyacrylamide and running conditions? How much primer should I load into the gel? Any help or other suggestions are greatly appreciated!
Save the hassle. Get new primers. It will cost $10
Hi tap14. I think you missed these two key parts of my message: "but I am in Peru and this all that I have to work with for now" & " I need to show to the vendor that they are degraded." If I was in US or other 1st world country I would have ordered a new set a few weeks ago. But things work a little differently here. So hopefully someone can offer some help as I need proof to get a refund as unfortunately they cost a little bit more here than $10. Thanks!
Hi tap14. I think you missed these two key parts of my message: "but I am in Peru and this all that I have to work with for now" & " I need to show to the vendor that they are degraded." If I was in US or other 1st world country I would have ordered a new set a few weeks ago. But things work a little differently here. So hopefully someone can offer some help as I need proof to get a refund as unfortunately they cost a little bit more here than $10. Thanks!
get some RNA that you are sure of and try again. if that works fine your primers are the problem if not then something else is the problem . good luck! how i wish i was in peru right now
Hi tap14. I think you missed these two key parts of my message: "but I am in Peru and this all that I have to work with for now" & " I need to show to the vendor that they are degraded." If I was in US or other 1st world country I would have ordered a new set a few weeks ago. But things work a little differently here. So hopefully someone can offer some help as I need proof to get a refund as unfortunately they cost a little bit more here than $10. Thanks!
Mass spect......if there are ESI-MS or MALDI.........that is the most simple method......but they should be high resolution one.......
Mass spect......if there are ESI-MS or MALDI.........that is the most simple method......but they should be high resolution one.......
[/quote]
Yes no mass spec here either, but the ceviche is delicious. Need some low-tech solutions. That is why I was asking about the polyacrylamide gel suggestion. Gracias para todos! 