36 replies to this topic

[yean_ny_nie]

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..

[T C]

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   .

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]

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   .

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..

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?

[phage434]

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.

[yean_ny_nie]

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.  

Edited by yean_ny_nie, 08 April 2009 - 08:05 PM.

[T C]

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.  

[yean_ny_nie]

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.  

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

[phage434]

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

[HomeBrew]

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

[yean_ny_nie]

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.  

[parkerrhm]

site directed mutagenesis kit is classified by vector size. it seems >6kb or <=6kb?! what size is your vector?

[yean_ny_nie]

parkerrhm, on Apr 10 2009, 11:44 AM, said:

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??

[chijing]

yean_ny_nie, on Apr 10 2009, 01:06 PM, said:

parkerrhm, on Apr 10 2009, 11:44 AM, said:

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.

Attached Files

•  21st_april_sdm_pcr_product_before_dpn1_digestion.bmp   2.25MB   179 downloads

[Kami23]

chijing, on Apr 22 2009, 07:00 AM, said:

yean_ny_nie, on Apr 10 2009, 01:06 PM, said:

parkerrhm, on Apr 10 2009, 11:44 AM, said:

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.

We use eppendorfs and they work very well the NYZ broth isnt a problem as we use SOC media (I dont know if this has something LB doesnt but maybe someone will!)

Good luck youll get there eventually!

[yean_ny_nie]

We use eppendorfs and they work very well the NYZ broth isnt a problem as we use SOC media (I dont know if this has something LB doesnt but maybe someone will!)

Good luck youll get there eventually!
[/quote]

Thanks Enthusiast for your reply. I will try using the SOC medium instead of LB broth. However, the XL-1 blue supercompetent cells are running out. Hence, I have to substitute it with self-prepared supercompetent JM109, which we have tested its transformation efficiency using pUC18. The plates shows good competency, however, when trying it (supercompetent JM109) with my dpn1 digested pcr product, it turns out no colony. I wonder what could went wrong.

Anyone could help with this as I have been repeating the procedure for quite some time. Thanks lots!!