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Top : New Forum Archives (2009-): : Molecular Cloning

Problems with Stratagene Site Directed Mutagenesis - (Apr/08/2009 )

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

-parkerrhm-

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??

-yean_ny_nie-

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.

-chijing-

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!

-Kami23-

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!

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!!

-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!

yean_ny_nie on Apr 9 2009, 06:04 AM said:

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 on Apr 23 2009, 01:15 PM said:

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!

mastermi on Apr 23 2009, 10:48 PM said:

yean_ny_nie on Apr 9 2009, 06:04 AM said:

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.

You really use just 1 µl of 10x reaction buffer for 50 µl reaction volume? Or did you just write it down false?
That would explain why you get no PCR product.
If you are using 5 µl, and the PCR doesn't work after several trials (annealing can be done at 50°-55°C), design new primers. Sometimes a pair of primers doesn't work for Quik Change for whatever reason...

I just did battle with a mutagenesis reaction and finally won after three weeks...so maybe I can be of some help.

I used Stratagene's QuikChange Lightning kit to mutate 5nt in each of three locations. But because the three locations are so close together, I had to do them one at a time (what a pain). First one went perfectly (300+ colonies), second one was okay (8 colonies...100% with mutation), third one didn't work. I tried four times and finally got it after I called Stratagene for troubleshooting.

Here's what they said:
-double extension time (so for Lightning kit, it's 30 sec per kb...do 1 min per kb instead)
-Use 8% final concentration of QuikSolution (normal is 1.5uL....use 4.08uL to get 8% in 51uL)
-Transform 4uL of reaction (their data shows no loss of efficiency up to 4uL)
-dNTPs might be bad

For me, I did everything they asked...and found the dNTPs were the issue. The protocol says to aliquot dNTPs to prevent freeze/thaw. So I put 1uL into each well of a strip tube. Problem is every time I had to do a reaction, I had to pull out the strip tube and cut off one of the tubes for my experiment. In the meantime, that 1uL thawed.

The tech support people do not know the formulation of the proprietary dNTPs, but know it is much higher than the usual 10mM. So it was suggested that I try using my own dNTPs and to try adding a lot more than I would for a normal PCR.

So I usually add 1uL of 10mM dNTPs for a 50uL PCR reaction. For the QuikChange reaction, I added 3uL of the 10mM.

I did two reactions identical in every way except for the dNTPs. In the end, the reaction with the kit's dNTPs had 0 colonies while my custom dNTP blend yielded about 20. 7 out of 8 clones I chose had the mutation.

I would try even more....maybe even 5-7uL.