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Problems with Stratagene Site Directed Mutagenesis - (Apr/07/2009 )

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I've planned to create single amino acid mutants for the alpha synuclein (A30P, A53T and E46K) using the Stratagene kit. As usual, I follow the protocol given, however it does not turn out well. No colonies were observed after the transfomation :wacko: .

I suspect there is a problem with the primer design as they are not up to 78oC as the Tm. Is there any other way to increase the Tm? As an additional information, I've diluted the primer with TE buffer.

I need help!! :( Thanks thanks..

-yean_ny_nie-

Hey,

Did you run the reaction before and after Dpn I digestion on the gel ?

The reaction isn't happening or there is some other problem?

Best,
TC

yean_ny_nie on Apr 8 2009, 10:44 AM said:

I've planned to create single amino acid mutants for the alpha synuclein (A30P, A53T and E46K) using the Stratagene kit. As usual, I follow the protocol given, however it does not turn out well. No colonies were observed after the transfomation :wacko: .

I suspect there is a problem with the primer design as they are not up to 78oC as the Tm. Is there any other way to increase the Tm? As an additional information, I've diluted the primer with TE buffer.

I need help!! :( Thanks thanks..

-T C-

T C on Apr 8 2009, 03:04 PM said:

Hey,

Did you run the reaction before and after Dpn I digestion on the gel ?

The reaction isn't happening or there is some other problem?

Best,
TC

yean_ny_nie on Apr 8 2009, 10:44 AM said:

I've planned to create single amino acid mutants for the alpha synuclein (A30P, A53T and E46K) using the Stratagene kit. As usual, I follow the protocol given, however it does not turn out well. No colonies were observed after the transfomation :P .

I suspect there is a problem with the primer design as they are not up to 78oC as the Tm. Is there any other way to increase the Tm? As an additional information, I've diluted the primer with TE buffer.

I need help!! :lol: Thanks thanks..



Ya, I've run gel for both before and after dpn1 digestion. However, there is no band appear on the gel. The control for both PCR reaction and transformation are working well. I got colonies for both controls. So, could it be the problems with the primer?

-yean_ny_nie-

Your PCR is not working. Try lowering your annealing temperature. Make sure your extension time is sufficiently long. After a day or so, redesign your primers.

Make sure you have sufficient template if you are doing only the recommended number of cycles. This is one of the few times when the amount of template can matter in a PCR reaction, since you are trying to avoid over-amplification of the plasmid, and need sufficient template so that few cycles will give sufficient product.

-phage434-

phage434 on Apr 9 2009, 10:52 AM said:

Your PCR is not working. Try lowering your annealing temperature. Make sure your extension time is sufficiently long. After a day or so, redesign your primers.

Make sure you have sufficient template if you are doing only the recommended number of cycles. This is one of the few times when the amount of template can matter in a PCR reaction, since you are trying to avoid over-amplification of the plasmid, and need sufficient template so that few cycles will give sufficient product.


I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help. :P

-yean_ny_nie-

Hey,

You forgot to add the enzyme or forgot to write it ? :lol:

Best,
TC

yean_ny_nie on Apr 9 2009, 09:34 AM said:

phage434 on Apr 9 2009, 10:52 AM said:

Your PCR is not working. Try lowering your annealing temperature. Make sure your extension time is sufficiently long. After a day or so, redesign your primers.

Make sure you have sufficient template if you are doing only the recommended number of cycles. This is one of the few times when the amount of template can matter in a PCR reaction, since you are trying to avoid over-amplification of the plasmid, and need sufficient template so that few cycles will give sufficient product.


I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help. :P

-T C-

T C on Apr 9 2009, 01:58 PM said:

Hey,

You forgot to add the enzyme or forgot to write it ? :)

Best,
TC

yean_ny_nie on Apr 9 2009, 09:34 AM said:

phage434 on Apr 9 2009, 10:52 AM said:

Your PCR is not working. Try lowering your annealing temperature. Make sure your extension time is sufficiently long. After a day or so, redesign your primers.

Make sure you have sufficient template if you are doing only the recommended number of cycles. This is one of the few times when the amount of template can matter in a PCR reaction, since you are trying to avoid over-amplification of the plasmid, and need sufficient template so that few cycles will give sufficient product.


I prepare the sample reaction as below:
1ul of 10 reaction buffer
1ul (25ng) of 25ug/ml dsDNA template
1ul (125ng) of 125ug/ml forward primer
1ul (125ng) of 125ug/ml reverse primer
1ul of dNTP mix
Top up to 50ul with dH2O

The cycling parameter are as below:
i) 95oC for 30secs
ii) 95oC for 30secs
iii) 55oC for 1min
iv) 68oC for 1min/kb of plasmid length (mine would be 6mins)
(ii) to (iv) is for 16 cycles.

I wonder where goes wrong. Please help. :blink:



opps.. sorry.. ya i forgot to write in.. ;)
1 ul (2.5U/ul) of PfuUltra HF DNA polymerase were added to the sample reaction.

-yean_ny_nie-

Anything special about the plasmid sequence? Extreme GC either low or high? Repeats? How was the primer designed?

-phage434-

We've had good luck with this kit. Did you use the QuikChange primer design program that Stratagene provides to design your primers?

-HomeBrew-

Ya, I'm using Stratagene’s web-based QuikChange® Primer Design Program to design the primers and the sequences are as below:

Primer sequences:
Primer Name Primer Sequence (5' to 3')
A30P 5'-gggtgtggcagaagcaccaggaaagacaaaaga-3'
A30P_antisense 5'-tcttttgtctttcctggtgcttctgccacaccc-3'
E46K 5'-ggctccaaaaccaagaagggagtggtgcatg-3'
E46K_antisense 5'-catgcaccactcccttcttggttttggagcc-3'
A53T 5'-gagtggtgcatggtgtgacgacagtggctgagaagac-3'
A53T_antisense 5'-gtcttctcagccactgtcgtcacaccatgcaccactc-3'


Oligonucleotide information:
Primer Name Length (nt.) Tm Duplex Energy at 68°C Energy Cost of Mismatches
A30P 33 79.14°C -46.84 kcal/mole 4.7%
A30P_antisense 33 79.14°C -40.31 kcal/mole 7.5%
E46K 31 78.98°C -45.00 kcal/mole 8.3%
E46K_antisense 31 78.98°C -40.86 kcal/mole 9.8%
A53T 37 80.01°C -49.06 kcal/mole 10.6%
A53T_antisense 37 80.01°C -50.76 kcal/mole 9.8%

However, the Tm of the primers delivered to us are not up to 78°C. They were just averagely at 60°C. ;)

-yean_ny_nie-
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