Hi,
We are using published primers for PCR amplification of HN gene of mesogenic NDV (Forward A TGG ACA GCG CAG TTA GCC AAG & Reverse GGT AGC CCA GTT AAT TTC CA)- 2090bp product size. We use vaccine R2B strain as positive control and initially, we were getting amplification at the expected size earlier and now we are getting smearing pattern in the field isolate. Please suggest me the solutions
PCR protocol
98-3mins
98-10 sec (35 cycles)
61.5-15 sec
72-1min 10 sec
72-7 mins
What are the changes I done in pcr
Gradient temperature
2 step cycle (10,30),single cycle (35).
Master mix (prime star master mix).
2 set of primer
Forward primer directly used during cDNA synthesis instead Random primer
Neat cDNA, 1:10 diution of cDNA.various dilutions of cDNA
Re amplifying the product
Dear karthik,
what is the method for genome extraction? do you check the integrity of genome?
cDNA kit- Thermo Scientific
RNA extraction by Trizol method and not by kit
Do you check the integrity of RNA on the gel? if it possible let me to see the picture of RNA?
reasonable suggestions about RNA extraction quality here. After total RNA extraction with Trizol run agarose RNA gel electrophoresis in denaturating conditions to see RNA integrity (works if you have rather fresh tissue samples). Also you can try INDICAL (former Qiagen) cador pathogen mini kit for RNA viral extraction -- that one is VERY good.
Also verify primers in BLAST, how really specific they are.
wish you good luck!
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