11 replies to this topic

[Curtis]

I'm trying to amplify a gene but I have problem with designing primer for the c-terminal of it.

the gene has a low GC content near the stop codon and that is why it is making this huge gap between the Tm of the two primers.

the gene reads as:

         1 atggactcat ccagggcaat cgggctgtac tttgattctg cccttccctc cagcagccta
       61 ttagcatttc cgatcgtcct acaagacaca ggagatggga agaagcaaat caccccacaa
      121 tacaggatcc agcgtcttga ctcgtggaca gacagtaaag aggattcggt attcatcacc
      181 acctatggat tcatcttcca agttgggaat gaggaagtca ctgtcggcat gattaatgat
      241 aatcccgggc acgagttact ttcctctgca atgctttgcc taggaagtgt cccgaacgac
      301 ggtgatcttg ttgagctggc gagggcctgc ctcactatgg tggtaacttg caagaagagt
      361 gcaactaaca ctgagagaat agtcttctcg gtagtgcagg cgccccgagt gctgcaaagc
      421 tgtatggtcg tggccaatag gtactcatca gtgaatgcag tgaaccatgt gaaagcacca
      481 gagaagatcc ctgggagcgg aaccctagag tataaggtga actttgtctc tttgactgtg
      541 gtgccgagga aggatgtcta caggatccca accgcagctt tgaaaagtat ctggctcaag
      601 cctgtacaat cttgcgctca atgtcactat gattgtggag gtggacccga agagcccgtt
      661 agtcaaatcc ctttccaagt cgatagttgg atactatgca attcttttct tgcatatcgg
      721 ggtatggtcc actgtagaaa ggaagggaaa gaaagtgaca ttgaccaagc tagaggggaa
      781 gataaggaga ctcaatctat ctgtcgggct caggattgtg ctcggacctt ccgtgcttgt
      841 gaaggcgaga ggtgcacgga caaggctgtt ggcacctttc ttctcagcca gtgggacagc
      901 ctgctatcct atagcaaatg cctctcccca ggtggtaaga tactctggag tcaaactgca
      961 cacctgcgga gtgtaaaaat tgtcattcaa gcaggcaccc aacgtgctgt cgcagtgacc
     1021 gctgatcatg aggttacctc taccaagata gagaagaggc ataccattgc taaatacaac
     1081 cctttcaaaa aatag

Forward: ATG GAC TCA TCC AGG GCA  LENGTH: 18 GC CONTENT: 55.6 % MELT TEMP: 56.0 ºC

Reverse: TTT TTT GAA AGG GTT GTA    LENGTH: 18 GC CONTENT: 27.8 % MELT TEMP: 43.9 ºC

what to do?

[phage434]

Make the second primer longer, TTT TTT GAA AGG GTT GTA TTT AGC

[Curtis]

phage434, on Dec 28 2009, 04:15 AM, said:

Make the second primer longer, TTT TTT GAA AGG GTT GTA TTT AGC

ok, thank you, as I thought. I tried not to add the gene's last TAG stop codon because I'm going to clone it to a EGFP plasmid's open reading frame. I wonder if this is still considered a complete gene or not ! I can't have the ending stop codon if I want to tag the gene. I hope my boss accepts it.

[microgirl]

Primers are cheap - try it with those two primers using 43deg as your annealing temp. Maybe you'll get lucky and only get one band or get only a couple well-separated bands.

Curtis, on Dec 28 2009, 02:08 AM, said:

I'm trying to amplify a gene but I have problem with designing primer for the c-terminal of it.

the gene has a low GC content near the stop codon and that is why it is making this huge gap between the Tm of the two primers.

the gene reads as:

         1 atggactcat ccagggcaat cgggctgtac tttgattctg cccttccctc cagcagccta
       61 ttagcatttc cgatcgtcct acaagacaca ggagatggga agaagcaaat caccccacaa
      121 tacaggatcc agcgtcttga ctcgtggaca gacagtaaag aggattcggt attcatcacc
      181 acctatggat tcatcttcca agttgggaat gaggaagtca ctgtcggcat gattaatgat
      241 aatcccgggc acgagttact ttcctctgca atgctttgcc taggaagtgt cccgaacgac
      301 ggtgatcttg ttgagctggc gagggcctgc ctcactatgg tggtaacttg caagaagagt
      361 gcaactaaca ctgagagaat agtcttctcg gtagtgcagg cgccccgagt gctgcaaagc
      421 tgtatggtcg tggccaatag gtactcatca gtgaatgcag tgaaccatgt gaaagcacca
      481 gagaagatcc ctgggagcgg aaccctagag tataaggtga actttgtctc tttgactgtg
      541 gtgccgagga aggatgtcta caggatccca accgcagctt tgaaaagtat ctggctcaag
      601 cctgtacaat cttgcgctca atgtcactat gattgtggag gtggacccga agagcccgtt
      661 agtcaaatcc ctttccaagt cgatagttgg atactatgca attcttttct tgcatatcgg
      721 ggtatggtcc actgtagaaa ggaagggaaa gaaagtgaca ttgaccaagc tagaggggaa
      781 gataaggaga ctcaatctat ctgtcgggct caggattgtg ctcggacctt ccgtgcttgt
      841 gaaggcgaga ggtgcacgga caaggctgtt ggcacctttc ttctcagcca gtgggacagc
      901 ctgctatcct atagcaaatg cctctcccca ggtggtaaga tactctggag tcaaactgca
      961 cacctgcgga gtgtaaaaat tgtcattcaa gcaggcaccc aacgtgctgt cgcagtgacc
     1021 gctgatcatg aggttacctc taccaagata gagaagaggc ataccattgc taaatacaac
     1081 cctttcaaaa aatag

Forward: ATG GAC TCA TCC AGG GCA  LENGTH: 18 GC CONTENT: 55.6 % MELT TEMP: 56.0 ºC

Reverse: TTT TTT GAA AGG GTT GTA    LENGTH: 18 GC CONTENT: 27.8 % MELT TEMP: 43.9 ºC

what to do?

[tea-test]

Curtis, on Dec 28 2009, 06:53 PM, said:

ok, thank you, as I thought. I tried not to add the gene's last TAG stop codon because I'm going to clone it to a EGFP plasmid's open reading frame. I wonder if this is still considered a complete gene or not ! I can't have the ending stop codon if I want to tag the gene. I hope my boss accepts it.

Well, then is it not possible to make it longer from the other end?

[HomeBrew]

Given your sequence, I would do something like:

WARNING: Right primer is unacceptable: Long poly-X

OLIGO			start  len	  tm	 gc%   any	3' seq 
LEFT PRIMER		  1   20   61.66   50.00  5.00  2.00 atggactcatccagggcaat
RIGHT PRIMER	  1092   25   60.25   28.00  5.00  2.00 ttttttgaaagggttgtatttagca
SEQUENCE SIZE: 1095
INCLUDED REGION SIZE: 1095

PRODUCT SIZE: 1092, PAIR ANY COMPL: 3.00, PAIR 3' COMPL: 1.00

The string of A's at the end of your sequence is a bit of a pain, but these should work OK despite the warning thrown...

[Curtis]

thank you guys,

I put the right primer at idtdna.com's calculator but it is giving me a very different Tm !


http://eu.idtdna.com...er/default.aspx

ttttttgaaagggttgtatttagca
MELT TEMP: 52.0 ºC

How come?

[phage434]

Because the calculations are empirical and not very good.  I pay little attention to the Tm calculations, and I think you should also.  Design primers 18-24 bp long, depending on the gc content.  Use an annealing temperature of 55 C, and things will usually work.  Try to make the 3' base a c or g.  Avoid long runs of the same base.  Avoid self priming and primer-dimers with 5' overhangs and exact matches at the 3' end.  Programs such as Primer3 work well if you have real problems.

[HomeBrew]

The primers I suggested were selected by Primer3 (version 1.1.4).  I agree with phage434 -- there are many ways to calculate the Tm of primers, and all produce different results.  We have used Primer3 to select thousands of primers, usually shooting for a calculated Tm of around 60C, and using them in PCR with an annealing temp of between 56C and 58C.  We have very few problems following these methods.

[Curtis]

guys,

I received the primers and I ran PCR with them. I also used controls for the target gene.

The thing is that I get a big smear above my band of 1kB.

the controls seem to be ok.

I tried reducing cDNA template, the number of cycles, elongation time etc. to reduce the smear, but it is still there after 1 week running PCR everyday.

I wanted to do PCR product purification but I decided to run all the products on gel and just cut the 1kB band.

Do you think this product would be suitable for cloning?

[HomeBrew]

Curtis, on Jan 23 2010, 07:32 AM, said:

Do you think this product would be suitable for cloning?

Probably -- it's tough to say without seeing the gel.  Can you post a picture of it?

Is the smear you're seeing well separated from the 1 kb band or is it contiguous with it?  How much amplification product did you get?

[Curtis]

sorry for the bad quality

forward primer CTTGGAATTCACCATGGACTCATCCAGGGCAATC
Tm=60.5

Reverse primer CTTGGTCGACTTTTTTGAAAGGGTTGTATTTAGC
Tm=55.7

and I put the annealing temperature at 51

I used 26 ng, 68 ng, and 1 ug of cDNA template but all give smear

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