10 replies to this topic

[grvsomani]

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.

Attached Thumbnails

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[Ameya P]

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]

grvsomani, on 14 April 2011 - 11:04 PM, 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?

[grvsomani]

hello GT

please find the attachment showing full detail of primers

Attached Files

•  New Microsoft Word Document.doc   37.5K   130 downloads

Edited by grvsomani, 15 April 2011 - 11:49 PM.

[grvsomani]

gt_ameya, on 14 April 2011 - 11:33 PM, 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.

[grvsomani]

hematopoietry, on 15 April 2011 - 01:00 AM, said:

grvsomani, on 14 April 2011 - 11:04 PM, 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

[Ameya P]

GRV,

Could you also tell us the conditions of the PCR cycle?

[grvsomani]

gt_ameya, on 15 April 2011 - 11:57 PM, 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.

Edited by grvsomani, 16 April 2011 - 06:48 AM.

[tea-test]

try finnzyme's phusion enzyme. with fermentas i got only smear, phusion worked perfect for full length gene amplification, especially the hot start enzyme.

[grvsomani]

tea-test, on 16 April 2011 - 08:06 AM, 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

[grvsomani]

grvsomani, on 17 April 2011 - 08:14 PM, said:

tea-test, on 16 April 2011 - 08:06 AM, 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

i have got long pcr taq enzyme from fermentas . i think it should work. give me ur opinion. what precautions should be taken for using it