smear in pcr +DNA polymerase mixture for long PCR - (Apr/15/2011 )
hello friends
i want to amplify 2.5 to 3.0 kb size gene through pcr. this gene have low copy number.i have applied all pcr troubleshooting for its amplification like change annealing time, magnesium con.,buffer, re synthesis primer, increasing no. of cycle, re amplification of PCR products, changes in RT protocol, using enhancer DMSO,Glycerol, Q-solution etc. Due to low copy number of this gene, i used to run 20 cycles of pcr using cDNA template ,without giving final extension step. then again i run 35 cycles of pcr using pcr product of 20 cycle. i have also tried 30 cycles then again 30 cycles then 35 cycles. generally i use 2ul of previous pcr as template. i always find smear in gel electophoresis. smear start from well to lower part.
please suggest me some solution.
now i m also thinking to use long PCR protocol. for this please suggest me how to prepare DNA polymerase enzyme mixture.i have taq polymerase LC (fermentas) and pfu (fermentas).
please suggest me in detail. i will be grate ful to u.
GRV,
Could you let us know the Tm of your primers and your PCR cycle please. I think, your PCR will work with a little bit of tweaking. 
grvsomani on Fri Apr 15 07:04:13 2011 said:
hello friends
i want to amplify 2.5 to 3.0 kb size gene through pcr. this gene have low copy number.i have applied all pcr troubleshooting for its amplification like change annealing time, magnesium con.,buffer, re synthesis primer, increasing no. of cycle, re amplification of PCR products, changes in RT protocol, using enhancer DMSO,Glycerol, Q-solution etc. Due to low copy number of this gene, i used to run 20 cycles of pcr using cDNA template ,without giving final extension step. then again i run 35 cycles of pcr using pcr product of 20 cycle. i have also tried 30 cycles then again 30 cycles then 35 cycles. generally i use 2ul of previous pcr as template. i always find smear in gel electophoresis. smear start from well to lower part.
please suggest me some solution.
now i m also thinking to use long PCR protocol. for this please suggest me how to prepare DNA polymerase enzyme mixture.i have taq polymerase LC (fermentas) and pfu (fermentas).
please suggest me in detail. i will be grate ful to u.
Is your negative control in the picture too?
hello GT
please find the attachment showing full detail of primers
gt_ameya on Fri Apr 15 07:33:37 2011 said:
GRV,
Could you let us know the Tm of your primers and your PCR cycle please. I think, your PCR will work with a little bit of tweaking.
hematopoietry on Fri Apr 15 09:00:50 2011 said:
grvsomani on Fri Apr 15 07:04:13 2011 said:
hello friends
i want to amplify 2.5 to 3.0 kb size gene through pcr. this gene have low copy number.i have applied all pcr troubleshooting for its amplification like change annealing time, magnesium con.,buffer, re synthesis primer, increasing no. of cycle, re amplification of PCR products, changes in RT protocol, using enhancer DMSO,Glycerol, Q-solution etc. Due to low copy number of this gene, i used to run 20 cycles of pcr using cDNA template ,without giving final extension step. then again i run 35 cycles of pcr using pcr product of 20 cycle. i have also tried 30 cycles then again 30 cycles then 35 cycles. generally i use 2ul of previous pcr as template. i always find smear in gel electophoresis. smear start from well to lower part.
please suggest me some solution.
now i m also thinking to use long PCR protocol. for this please suggest me how to prepare DNA polymerase enzyme mixture.i have taq polymerase LC (fermentas) and pfu (fermentas).
please suggest me in detail. i will be grate ful to u.
Is your negative control in the picture too?
there is no negative control in the picture. actually on left extream and middle have marker double digest. other are pcr products
GRV,
Could you also tell us the conditions of the PCR cycle?
gt_ameya on Sat Apr 16 07:57:53 2011 said:
GRV,
Could you also tell us the conditions of the PCR cycle?
94'c = 5 min
94'c = 1 min
52'c = 45 sec
72'c = 2 min 30 sec
72'c = 5 min
4'c = hold
Due to low copy number of this gene, i used to run 20 cycles of pcr using cDNA template ,without giving final extension step. then again i run 35 cycles of pcr using pcr product of 20 cycle. i have also tried 30 cycles then again 30 cycles then 35 cycles. generally i use 2ul of previous pcr as template. i always find smear in gel electophoresis. smear start from well to lower part.
try finnzyme's phusion enzyme. with fermentas i got only smear, phusion worked perfect for full length gene amplification, especially the hot start enzyme.
tea-test on Sat Apr 16 16:06:13 2011 said:
try finnzyme's phusion enzyme. with fermentas i got only smear, phusion worked perfect for full length gene amplification, especially the hot start enzyme.
thanks