Help with PCR Please

[ff_fairy]

Can anyone help or advise me. I am a Student and have been trying to amplify cDNA produced from RNA unsuccessfully for a number of weeks now in order to complete a gene sequence where I have a small gap for around 40 nucleotides.

I performed the RT-PCR First Strand Synthesis using Fermatas Revert Aid Firrst Strand cDNA Synthesis Kit and after 10 attempts got a band using the following:

12ul MyTaq

1ul 18s Forward Primer

1ul 18s Reverse Primer

1ul concentrated cDNA

9.5ul Nuclease Free H2O

 

This was to check there was actually cDNA produced.

Primers were then designed from either side of the "missing segment" of around 45 nucleotides.

 

To amplify the gene in question (POR Gene) I have tried several things.

Initially I used:

16.25ul Nuclease Free H20

5ul Buffer

0.5ul dNTPs

1ul Fwd Primer

1ul Rev Primer

1ul Template

0.25ul Enzyme (Phusion Hotstart II)

 

PCR conditions used were

1 cycle:

98oC for 30 seconds

39 cycles of:

98oC 10 seconds

54oC 30 seconds

72oC 30 seconds

1 cycle:

72oC 5 minutes

 

I have now tried altering the following:

Changing to Velocity Enzyme

Seeded PCR

Halving the enzyme amount

Reducing extension time

Changing annealing temp from 54oC to 50oC and also 45oC

Adding more Template

Increasing annealing time to 60 seconds

 

All of the above has produced no bands at all, or smears, or the PCR product still in the wells after gel was ran.

 

I am really stumped now and would appreciate some advice.

I hope I have included everything I need in this post.

 

Many Thanks in advance.

[jp255]

I am assuming you tested the primers and know for sure that they work.

 

 

Try lowering the extension temperature? That cured the problem I had not too long ago. I was trying to PCR a fragment which was particularly AT rich, at higher extension temperatures I think that this AT rich part melted and didn't amplify. Extension worked at 58 in the end

[Arete]

I am assuming you tested the primers and know for sure that they work.

 

 

Try lowering the extension temperature? That cured the problem I had not too long ago. I was trying to PCR a fragment which was particularly AT rich, at higher extension temperatures I think that this AT rich part melted and didn't amplify. Extension worked at 58 in the end

 

I was going to suggest a touchdown profile. Start at 50C and work up to 60C.

 

Have you adjusted the salt concentration? a salt gradient would be my net move if the tdPCR failed.

[ff_fairy]

I will try the touchdown PCR thank you for your suggestions.

 

I have not adjusted the salt concentration at all no.

 

I will try the touchdown PCR thank you for your suggestions.

 

I have not adjusted the salt concentration at all no.