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cloning through PCR - (Apr/02/2007 )

Can any body tell me how to clone in a way that red part of gene is replaced by black part through PCR method. Tootal gene is around 2.9 kb and coloured part is 900 bp around.

-samita-


My suggestion for a PCR strategy is as below

Making the templates
Make the above primers 1, 2, 3, and 4
All primers must have the same melting temparture.
Importantly Primer 2 is composed of 2 halves. Both halves of the primer has the same tm. Ie the black half binds to the black DNA fragment with the tm of 58 Celsius. the red half binds to red DNA segment with a tm of 58 Celsius

Amplify black DNA fragment with primers 1 and 2 - this is call vial 1
Amplify red DNA fragment with primers 3 and 4 - this is call vial 2

Run PCR reaction about 20 cycles using a proof reading high fidelity polymerase like KOD hifi.
Do not under any circumtance use taq. Your PCR product is far too large and Taq makes too many mistakes.

Making single stranded DNA by asymertic PCR
Once the reaction is complete, take 5ul and use it as a template for a 50ul PCR mix. THis 50ul reaction will be an asymetric reaction, where there will be only one (1) primer is present in the reaction. You should use double the amount of primers usually used. Do this for each vial type.

eg
Take 5ul of vial 1, use it as a template. The single primer added to the reaction is primer 1. This reaction mix will be call vial 3

In a saperate reaction, take 5ul of vial 2 and again use it as a template for a 50ul PCR reaction. Use primer 4 as the single primer for the reaction. This PCR mixture will be called vial 4

Conduct the PCR reaction for 10 cycles

Fusing the single standard DNA
The mix 25ul of vial 3 and 25ul of vial 4 together. Run the PCR reaction for a futher 15 cycles.

Once the reaction is complete you should have your PCR product. I would advice that you engineer restriction sites onto primer 1 and 4, so that you can ligate the final product into your vector.

Remember to add enough bp around the restriction site. A restriction site should by default have 6bp on either side. (Although several enzyme will work with less, 6 is a nice number) Always check the number of bp that are required to flank a restriction site for the enzyme to cut it. The enzyme does have a foot print, it need to stand on something to cut the site.

Once you have the fusion product in a plasmid. Scene by restriction digest. Then sequence 3 to 4 isolates to make sure there are no mistakes in the product.

-perneseblue-

First of all thanks for the reply and suggestion and it will be a very good to do that. I have one more idea which i attached the picture to make two overlapping PCR. My question with your strategy and my attached picture is that in Primer 2 in your picture and primer with circle in my pictre have homology with other fragment to which gene is fused.
in your picture primer 2 is reverse primer (Black part) i will take sequece make complementary and reverse it its black part but the red part is what its also complementary and reverse or just complemenraty part of the other gene whihc has to be fused.
Similarly my attached picture the circle part of the reverse primer and forward primer it should be reverse complemntary or just complementary part of the other gene which has to be fused.
I shall be very thanksful to you.
regards


QUOTE (perneseblue @ Apr 2 2007, 04:35 PM)

My suggestion for a PCR strategy is as below

Making the templates
Make the above primers 1, 2, 3, and 4
All primers must have the same melting temparture.
Importantly Primer 2 is composed of 2 halves. Both halves of the primer has the same tm. Ie the black half binds to the black DNA fragment with the tm of 58 Celsius. the red half binds to red DNA segment with a tm of 58 Celsius

Amplify black DNA fragment with primers 1 and 2 - this is call vial 1
Amplify red DNA fragment with primers 3 and 4 - this is call vial 2

Run PCR reaction about 20 cycles using a proof reading high fidelity polymerase like KOD hifi.
Do not under any circumtance use taq. Your PCR product is far too large and Taq makes too many mistakes.

Making single stranded DNA by asymertic PCR
Once the reaction is complete, take 5ul and use it as a template for a 50ul PCR mix. THis 50ul reaction will be an asymetric reaction, where there will be only one (1) primer is present in the reaction. You should use double the amount of primers usually used. Do this for each vial type.

eg
Take 5ul of vial 1, use it as a template. The single primer added to the reaction is primer 1. This reaction mix will be call vial 3

In a saperate reaction, take 5ul of vial 2 and again use it as a template for a 50ul PCR reaction. Use primer 4 as the single primer for the reaction. This PCR mixture will be called vial 4

Conduct the PCR reaction for 10 cycles

Fusing the single standard DNA
The mix 25ul of vial 3 and 25ul of vial 4 together. Run the PCR reaction for a futher 15 cycles.

Once the reaction is complete you should have your PCR product. I would advice that you engineer restriction sites onto primer 1 and 4, so that you can ligate the final product into your vector.

Remember to add enough bp around the restriction site. A restriction site should by default have 6bp on either side. (Although several enzyme will work with less, 6 is a nice number) Always check the number of bp that are required to flank a restriction site for the enzyme to cut it. The enzyme does have a foot print, it need to stand on something to cut the site.

Once you have the fusion product in a plasmid. Scene by restriction digest. Then sequence 3 to 4 isolates to make sure there are no mistakes in the product.

-samita-




Here is my revision of the above strategy. If you add primers 1 and 4 into the second PCR reaction, your yields should improve. There is no real need to add homology sequence to primer 3. But if you do add homology sequence to both primer 2 and 3, try to keep it short, if the overlap becomes too big, proper annealing between the two templates will be difficult.(tm becomes too high)

-perneseblue-