Dear Nadine! First of all, if you use pfu polymerase, use 2 min of elongation per 1 kb, so 6 min for 3 kb fragment. Second thing, which is important for cloning: pfu polymerase produces blunt-ended fragments, so TA cloning vector will be unsufficient. The best way: prepare blunt-ended dephosphorylated vector (cut with SmaI, EcoRV etc. followed by alcaline phosphotase treatment) and kinase oligos before PCR (because synthetic oligonucleotides do not have 5'-phosphates)

Dear Nadine, Sorry i cannot view your answer from my dashboard.

Dear Alexey, I used 3 min. (1 min. for 1 kb) of elongation time, but you recommend 2 min. for each kb to be amplified. Please find my reaction conditions for a gradient PCR reaction with pfu polymerase and suggest any changes:

Pre-denaturation: 94oC for 5 min.

Denaturation: 94oC for 1 min

Annealing: 45-65oC for 1 min

Extension: 72oC for 6 min

Go to 2- 35 times

Final extension- 72oC for 10 min

Regarding cloning, we used to make sticky-ends of blunt-ended fragments by PCR product refresh, followed by TA cloning. I would like to know whether a 3 kb size insert can be efficiently cloned into a TA vector after amplification and purification.

Thank you

I advise using Platinum Taq DNA Polymerase High Fidelity from Invitrogen with next PCR conditions:

- 1 cycle 94C for 3 min,

- 40 cycles

94C 30 sec, primer Tm 1 min, 68C 3:30 min

-1 cycle 68C 10 min, 4C

You could try Roche's Long Range Expand kit for Long range PCR! Good luck!

The most important steps are:

1. Proof reading Taq polymerase to be used i.g. PFU and others

3. elongation step to at 72C for maxmum 30 Cycles for minimum 2 min and mximum 3.5 min

Hello, in the past I amplified segments of mtDNA of around 2700 bp using a standard Taq Polymerase and standard PCR conditions:

1) Initial denaturantion: 95°C for 5 minutes

2) Denaturation: 94°C for 40 seconds

3) Annealing: 52°C for 1 minute (duration and temperature of this step depend on your primers)

4) Extension: 72°C for 1.5 minutes

5) Final Extension: 72°C for 10 minutes

Steps from 2 to 4 were repeated 35 times.

As for cloning, pGEM®-T and pGEM®-T Easy Vector Systems have a good efficiency in the range 0.5 to 3 kbp.

I hope this can help.

Good luck,

Massimo

Hi,

There are several polymerase these days. A PCR to amplify 3kB should be easy nowadays. The elongation time is depending on the polymerase and template (genomic DNA, plasmid, etc.) you use, and Tm on primer set. For example the

Phusion® High-Fidelity DNA Polymerase from NEB will result in a high quality amplicon.

98-30s

98-30s

Tm-(10-30)s

72-(15-30)s/kB

(25-35)x

72-(5-10)min

4-----

To use this PCR product for TA cloning you will need to A-tail prior TA reaction. Because your insert is larger (3kB) your cloning yield will be considerably lower than when you use 1kB. But it should work.

See the NEB page for more information on cloning and PCR.

Cheers,

TS.

TA cloning vectors should be fine for cloning 3 kb fragments. As Massimo, I have used the pGEM®-T Systems and work fine for 3-4 kb fragments.

Regarding your PCR, have you tried reamplifying the PCR product?

Another option would be doing nested PCR (if you have enough sequence information).

Let me know if you would like me to expand on these suggestions.

Hope it helps,

Bàrbara

Thanks all. I have tried conducting a gradient PCR with elongation time of 3 min. But there is absolutely no amplification. As per your suggestion, i may extend it to 6 min with Taq DNA polymerase.

Dear Dr. Barbara, i have not re-amplified the PCR product or conducted nested PCR, although we have the full ORF of the gene in question, derived from genomic clone we need to confirm the sequence by cloning. So any suggestions on the same will be highly appreciated.

If you do not need the full fragment and just want to confirm the sequence I would suggest to amplify the region in two fragments instead of one. Make sure the two fragments overlap. Having smaller fragments should give less problems.

You may also change the external primers, sometimes just moving the primer a few bp modifies their yield significantly.

I understand your precious argument. But by improving the amplification efficiency (2-3 fragments), can we clone the entire gene for sequence confirmation. Basically, i need to confirm the entire 2.8 kbp ORF region and also conduct the RACE-PCR for both 5'- and 3'- ends.

Hi Bharat,

It would be helpful to know what your template material is. Also, how many cycle you are running. 3000 bp is long, but it shouldn't be out of reach with the appropriate enzyme and mastermix. 8000 bp has been achieved.

I think it would be worth trying to amplify some shorter segments using your present outer primers and some new internal primers just to check that the primers actually work.

You didn't say if you had used a primer-design program for primer design. Optimum primer design is always an issue when doing challenging amplifications. For example, the least tendency to primer-dimer formation will result in your long, hard-to-amplify PCR product being out-competed by easily amplified dimers.

Hope this helps

Andrew

I have been trying to amplify RFT1 gene that has size of ~534 bp. Initially I used Taq polymerase and was giving me multiple band when viewed using 1% gel.

Now I'm using pfu polymerase for proof-reading and conducting PCR with an extension time of 1.20 min. But no any amplification after a gradient PCR !

Please suggest (both Taq and pfu)?

Thanks