Site Directed Mutagenesis

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udvets
udvets's picture
Site Directed Mutagenesis

Hi People,
I hope somebody can help me with this.
I am trying to create two base pair mutation in one of the promoter. I have this promoter already inserted in the plasmid. Total size of plasmid is 5.7 kb. My primers are  44mer. Two base pair mutation is exactly in the middle of primers. I was using iProof taq from Biorad. Following is the protocol I am using.

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Ingredient

Amount to add to tube

5x HF buffer

5 ml

5x GC buffer

5 ml

10mM dNTP

1 ml

20mM F-primer

1.25 ml

20mM R-primer

1.25 ml

50 ng/ml DNA template

1 ml

Kit MgCl2

2.5 ml

Kit DMSO

1.5 ml

Enzyme

0.5 ml

Water

32.5 ml

Total

50 ml

and the cycle is as follows,

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Step

Temperature

Time

1 cycle of

1

98°C

3 minutes

30 cycles of

2

98°C

10 seconds

3

55°C

30 seconds

4

72°C

5 minutes

1 cycle of

6

72°C

10 minutes

7

8°C

forever

But i am not getting any success with this protocol. should i order different taq? Anyway also we are out of this taq so i may have to order different taq. can you suggest me if something better than this??
I am stuck with this mutagensis since long time ...Please help me out.
Thansk in advance...
Umesh

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Ivan Delgado
Ivan Delgado's picture
Hi Umesh,

Hi Umesh,
When you say this is not working, do you mean the PCR is not working or the clones you've sequenced do not contain the mutation you are looking for?
My suggestions would depend on your answer to this question, but in the mean time here are some things to consider:
1. Only introduce one mutation at a time in your primer (if your PCR is not working). In the past I introduced a ton of mutations into genes, and I did it one mutation at a time. Once you get one clone with one of the mutations, use that clone to add the second mutation.
2. If necessary, although not the best alternative, introduce the mutations into a smaller fragment (200-300 bp) which you can then clone into your gene. The larger the fragment you are trying to PCR the trickier it can get
3. Last recourse, spend some money. You can get a "large" DNA fragment synthesized, with your favorite restriction enzymes and all, and then clone it into your gene. Places like IDT will charge you about $300 for a 400 bp fragment with all the modifications you will ever need. And they send it to you cloned into a plasmid.
Hope this helped.

udvets
udvets's picture
Hi Ivan,

Hi Ivan,
Thank you very much for your reply. I really appreciate your help.
I am not getting my pcr worked actually. One of the option you gave me sounded better actually i.e. to introduce the single mutation and then once we got that clone successfully, introduce second mutation. I will definitly think about that. Meanwhile can you suggest me any particular high fidelity Taq from any company?
Thanks a lot
I really appreciate your help.
Thanks
Umesh

Jason King
Jason King's picture
What about using a SDM kit

What about using a SDM kit such us Quick-change? They work well.

Ivan Delgado
Ivan Delgado's picture
When it comes to high

When it comes to high fidelity Taqs, to be completely honest, a fair number of them out there are as good as the others. I've always believed in Pfu. My only suggestion is to use a mixture of a proofreading Taq like Pfu and another Taq, even a non-proof reading Taq like regular Taq. Proofreading Taqs are great at not introducing errors, but they are not very good at making that much DNA. By mixing the proof-reading Taq with a more processive Taq, you get the proof-reading activity of one while at the same time increasing your yield.

Jason King
Jason King's picture
The Quick-change kit is not a

The Quick-change kit is not a PCR based kit: See below:
 
http://www.stratagene.com/products/displayProduct.aspx?pid=505
 
Non-PCR Alternative Yields Greater than 80% Efficiency
The QuikChange® XL site-directed mutagenesis kit* is a fast, easy and efficient non-PCR alternative to other site-directed mutagenesis techniques. QuikChange XL uses a unique method that does not require single-stranded DNA or labor intensive, difficult-to-perform steps. Cesium chloride-purified or miniprep DNA, (such as plasmid DNA purified with the StrataPrep® plasmid miniprep purification kit) can be mutated and transformed with 80-100% of the resulting colonies containing the desired mutations.