12 replies to this topic

[dan_]

Hi all!

I want to make an overlapping PCR with 2 fragments, one has 2200bp and the other has 6000bp. The two fragment share 45 pb homology to their end.

I know that, theorically, I can generate a ''new'' fragment of 8200 bp, but I have no idea of the experimental condition that I should use to get succes! If someone can suggest me some experimental conditions, such as amont of DNA to put into my PCR, PCR amplification conditions and polymerase to use, it would be very helpfull.

thank!

[Curtis]

Guys, I have the same issue. I am aware this topic has been discussed many times here, but I still have problem finding the best protocol. There are some protocols on the website too, but ....I don't know which one to trust.  I want to design fragments of 3k and 4k in size and then run overlap-PCR. how many nucleotides the overlap regions should be? what would be the PCR condition?

[pDNA]

http://www.biotechni...ues-280116.html

all the best! ...also go for the supplementary!

Reagards,
p

[Curtis]

thanks, I think this thread needs to be moved to PCR subforum

[Evanescence]

at least 18bp but i prefer more...

you can use ratio 1:1 (template 1;template2)
for fusion pcr annealing temperature and extension time really critical

[dan_]

Thanks Evanescence for your helpful advice. But can you be more precise about pcr annealing temperature and extension time should I need to get succes? Tm of the homologous regions are approx 81oC (calculated with finnzyme tm oligonucleotide calculator).

regarding quantity of DNA template to put into the PCR reaction, I have read on Internet to put about 50ng of each fragment. Is it the amont of DNA you recommend me?

[Curtis]

Finnzyme? are you using Phusion? It usually has high Tm. some people don't recommend it for overlap PCR.

[Evanescence]

Sorry dan, i am late since i am not really well..
For me no need for you to calculate the Tm for overlapped region because the whole first fragment is like a forward primer to second template....what important here is your reverse Tm...try to do gradient temperature of annealing 5 0r 10 below the reverse primer..about the concentration i done successfully without checking the concentration of DNA but do compare them via gel only...somehow people also recommend to do some dilution...if you can google some protocol online like "oakley fusion.overlapped pcr" they did tell in detail how they done the fusion...but me, i do a bit different...

you know dan, sometimes, taq alone never give best result..i did mix two polymerase for good result..i use pfu and taq..mean you also need to mix thrir buffer together too...

for extension, it's depend on how long your fusion size....

[Evanescence]

Curtis, on 19 February 2012 - 08:52 AM, said:

Finnzyme? are you using Phusion? It usually has high Tm. some people don't recommend it for overlap PCR.

Hi Curtis,
For fusion pcr i never use both of them...it's expensive and i just use what my lab has...
I used to mix pfu polymerase and taq polymerase (mix their buffers too) and i got better amplification for fusion pcr
the reason is, pfu-high fidelity/proof reading and help the fragment to bind to specific overlapped region (but it slow)...while taq will
speed up the synthesis of new DNA in pcr..the combination is perfect for fusion..it's cheaper and work for me

all the best_Evanescence

[pDNA]

Phusion seems to be the only polymerase that works fine for overlap extension PCR (according to the paper referenced above) ...never used a different enzyme ...and if the paper is right ...i would not recommend to do so!

Regards,
p

[Evanescence]

pDNA, on 19 February 2012 - 11:11 AM, said:

Phusion seems to be the only polymerase that works fine for overlap extension PCR (according to the paper referenced above) ...never used a different enzyme ...and if the paper is right ...i would not recommend to do so!

Regards,
p

No, i never use phusion for overlapping pcr but i still got nice band,
as i told you using combination of polymerase of pfu and taq could help (for second pcr)
In addition to that, most of people recommend to pcr first fragment with pfu or vent polymerase
this is because the polymerase used here will not add poly A...

let say your gene: NNNNNNNNXXXXXXXXX + XXXXXXXXXXXJJJJJJJJJJJJJJJJJ
if you use some polymerase that produce A tails the first fragment will be AAAANNNNNNNNNXXXXXXXXXAAA

the A will interfere from bind to XXXXXXXXJJJJJJJJJJJJ but that's not critical....sometimes if you are lucky you still can have fusion pcr very well...but the chance is..................

[pDNA]

using a mix of polymerases is what they do for the Stratagene Quikchange kit ...where u have "short primers" (~30-60 nt) overlapping primers ...the method i referring to uses "megaprimers" ...so a whole fragment (>100 nt>) as a primer ...and it seems that here Phusion is the only polymerase that is able to yield sufficient amounts of colonies ...so we shouldn't mix protocols here and not confuse people.

Regards,
p

[Curtis]

here, see this
http://www.protocol-...or-overlap-pcr/