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Help with PCR and Gel Electrophoresis. Not getting any bands


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8 replies to this topic

#1 ff_fairy

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Posted 07 September 2012 - 06:00 AM


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.


#2 phage434

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Posted 07 September 2012 - 07:04 AM

How were your primers designed? Anything special about the template (high GC, low GC?, structure)?

#3 ff_fairy

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Posted 08 September 2012 - 12:52 AM

I already have 2 sequences, one is the 5' end of the gene, and the other the 3' end. There is however a gap of around 45 nucleotides in the middle which is what I am trying to amplify. The primers were designed from these sequences either side of the gap, are 20 nucleotides long and not particularly GC rich.

5'-CACGTCCTCCTTTGGACCAT-3'
5'-CGAGAAGGCGAGGTACAACT-3'

#4 phage434

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Posted 08 September 2012 - 11:16 AM

Those look like perfectly reasonable primers. How do you know the length of this gap? Are you able to amplify the known regions of your gene from the same template sample?
Perhaps the gap is really an intron, and you have been amplifying genomic DNA rather than cDNA.

#5 ff_fairy

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Posted 08 September 2012 - 03:12 PM

Did a Blast search and the compared the 2 sequences as from same family, using ClustalW2 and got an idea of the length of the gap.

#6 phage434

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Posted 08 September 2012 - 03:39 PM

And, can you amplify the portions you have sequence for from your cDNA template?
You could amplify genomic DNA with the two primers you have. If you get a short result, then you will cover the gap. If you get a long result, you will know there is an intron. What is the end goal? Are you trying to make the gene? If so, then you can probably substitute one of the homologous sequences you have apparently found for the 9 aa that are missing.

#7 ff_fairy

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Posted 09 September 2012 - 01:46 AM

The sequences I already have were given to us along with several others from another research dept and when we did a BLAST search of them all in the database, the two we have are the ones that came up as the POR Gene but there is a small sequence missing in between them.

#8 phage434

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Posted 09 September 2012 - 06:29 AM

So, I would recommend that you try to amplify the sequences you already know are present. This will check that your cDNA (or perhaps genomic DNA) really is present and of sufficient quality. I would also try dilutions of your template.

#9 ff_fairy

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Posted 10 September 2012 - 07:49 AM

I have already tried dilutions of the template, but will try the sequences I already know, thank you




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