
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!!

Posted 07 April 2009 - 09:14 PM
Posted 07 April 2009 - 11:04 PM
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
.
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..
Posted 08 April 2009 - 06:42 PM
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,
TCI'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
.
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..
Posted 08 April 2009 - 06:52 PM
Posted 08 April 2009 - 08:04 PM
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.
Edited by yean_ny_nie, 08 April 2009 - 08:05 PM.
Posted 08 April 2009 - 09:58 PM
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.
Posted 09 April 2009 - 04:58 PM
Hey,
You forgot to add the enzyme or forgot to write it ?
Best,
TCYour 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.
Posted 09 April 2009 - 06:25 PM
Posted 09 April 2009 - 07:04 PM
Posted 09 April 2009 - 07:44 PM
Posted 09 April 2009 - 09:06 PM
site directed mutagenesis kit is classified by vector size. it seems >6kb or <=6kb?! what size is your vector?
Posted 21 April 2009 - 10:00 PM
site directed mutagenesis kit is classified by vector size. it seems >6kb or <=6kb?! what size is your vector?
my vector backbone size is 4.4kb while the insert will be 1.5kb. I assumed it would be total of 5.9kb.
Posted 22 April 2009 - 08:19 AM
site directed mutagenesis kit is classified by vector size. it seems >6kb or <=6kb?! what size is your vector?
my vector backbone size is 4.4kb while the insert will be 1.5kb. I assumed it would be total of 5.9kb.
If I got this band (as indicated on the gel picture) after pcr amplification as below, does it mean that my site-directed mutagenesis should be successful? Is it okay if I use other supercompetent cells (e.g. self-prepared JM109 supercompetent cells) instead of the Xl-1 blue supercompetent cells provided by Stratagene?
By the way, I'm using LB Broth instead of the recommended NYZ broth during the cells' growth as our lab does not provide us with the latter one. Could this affect the transformation efficiency as well?
During the heat shock procedure, we are using 1ml eppendorf tube instead of the 14ml falcon round-bottom tube as my supervisor didn't order any. Is there anyone try using eppendorf tube and was successful?
Thanks lot.
Posted 23 April 2009 - 04:15 AM
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