Determine annealing temperature of primers - (Apr/26/2009 )
ah6tyfour on May 6 2009, 09:06 AM said:
Phusion will take care of both =)
ya i had tried a range from 56 to 62 but i didnt got any ampilfiacation at any mgcl2 conc......
so i shifted to further low annealing temp.........but at 50 i got smear ..........nw wat ????
Did you design your primers using some sort of program like Primer3? If you designed them by hand, you might want to make sure you are ordering the correct thing. I've had many a student spend a month on a troublesome PCR only to realize there was a mistake in the sequence of the antisense primer.
Also, did you BLAT your primers with Genome Browser to make sure they're specific? Also use Genome Browser's PCR function so you can paste in your sense and antisense primers and have it predict the product. Make sure the product is the right size and in the right region of the genome.
And what is your template? Are you sure it's good? I've had PCRs fail because the DNA went out of solution so I ended up diluting it way too much (like gDNA at 600ng/uL until it all spindled together). What liquid is your template in and what volume of template are you adding? If your DNA is in TE and you are adding a lot of it, you might inhibit the reaction. Same with cDNA from an RT reaction. Add more than 10% of your PCR volume and the salt from the cDNA will start messing up your PCR.
You can also try Touchdown PCR. The annealing temp will increase a little bit each cycle for the first 10 cycles or so....and the hope is that you'll get enough amplified at some temp that it can start acting as good template.
If everything checks out and you are getting nothing, use Phusion Polymerase....you will get it.
ah6tyfour on May 7 2009, 12:25 AM said:
Also, did you BLAT your primers with Genome Browser to make sure they're specific? Also use Genome Browser's PCR function so you can paste in your sense and antisense primers and have it predict the product. Make sure the product is the right size and in the right region of the genome.
And what is your template? Are you sure it's good? I've had PCRs fail because the DNA went out of solution so I ended up diluting it way too much (like gDNA at 600ng/uL until it all spindled together). What liquid is your template in and what volume of template are you adding? If your DNA is in TE and you are adding a lot of it, you might inhibit the reaction. Same with cDNA from an RT reaction. Add more than 10% of your PCR volume and the salt from the cDNA will start messing up your PCR.
You can also try Touchdown PCR. The annealing temp will increase a little bit each cycle for the first 10 cycles or so....and the hope is that you'll get enough amplified at some temp that it can start acting as good template.
If everything checks out and you are getting nothing, use Phusion Polymerase....you will get it.
yah i had designed my primers by primer3 only....
and yes i had checked their specificity too and both sense and antiensse match well.......
m using genomic DNA as my template and it is in TE ....
Redesign and resynthesise. 2(A+T) + 4(G+C).
swanny on May 11 2009, 12:23 AM said:
thanks a lot 4 advise