11 replies to this topic

[zack]

hi admin and chatter,

i had a problem with my primer design. pls refer to my gel picture. no 1 and no 2 is not my band actually,  but no3 is my band. my question are

1. how to remove another unspecified  band (no 1 and no 2).
2. how possible make single band on my gel



if u had same problem with me or u had some experience pls give some opinion and how to solve the problem..


this is PCR product

the PCR conditions are

Intial Denaturation 98C - 5 min

total 35 cycles

denaturation - 94C - 1min
annealing - 50 - 60C (gradient, u can see at the gel picture above) - 1 min
extension 72C 2 min

Final Extension - 72C

no 3 on the gel picture is ~272 bp

thank you all.

Edited by zack, 12 February 2009 - 09:09 AM.

[scolix]

What bands r these? PCR products ???

[WHR]

Hi,
Your pcr condition is not optimized. PCR normally will produce a huge amount of product, regardless of the specificity.

[perneseblue]

could you write down the PCR conditions that you are using? And what polymerase are you using?

If band 3 is the desired PCR product, I suggest that your reduce extension time. How big is band3?

You might also want to raise annealing temperature a few degrees.

[little mouse]

could it be genomic DNA (plus intron) ?
please give more details

[zack]

little mouse, on Feb 12 2009, 07:42 AM, said:

could it be genomic DNA (plus intron) ?
please give more details

this is PCR product

the PCR conditions are

Intial Denaturation 98C - 5 min

total 35 cycles

denaturation - 94C - 1min
annealing - 50 - 60C (gradient, u can see at the gel picture above) - 1 min
extension 72C 2 min

Final Extension - 72C

no 3 on the gel picture is ~272 bp

[perneseblue]

you can drop the annealing time to 30sec. 1min is very long. Depending on the complexity of your template (is it genomic DNA? plasmid DNA?) can you probably go lower.

extension time is too long. Read the user manual that describes your polymerase. I would guess, dropping the extension time down to 30sec would be helpful. It can probably go lower.

[zack]

perneseblue, on Feb 12 2009, 09:25 AM, said:

you can drop the annealing time to 30sec. 1min is very long. Depending on the complexity of your template (is it genomic DNA? plasmid DNA?) can you probably go lower.

extension time is too long. Read the user manual that describes your polymerase. I would guess, dropping the extension time down to 30sec would be helpful. It can probably go lower.

yea.. this is genomic DNA... Read the user manual that describes your polymerase? u mean taq polymerase?

Edited by zack, 12 February 2009 - 09:35 AM.

[perneseblue]

zack, on Feb 12 2009, 09:27 AM, said:

perneseblue, on Feb 12 2009, 09:25 AM, said:

you can drop the annealing time to 30sec. 1min is very long. Depending on the complexity of your template (is it genomic DNA? plasmid DNA?) can you probably go lower.

extension time is too long. Read the user manual that describes your polymerase. I would guess, dropping the extension time down to 30sec would be helpful. It can probably go lower.

yea.. this is genomic DNA... Read the user manual that describes your polymerase? u mean taq polymerase?

There are many types of polymerase on the market. With some of the high fidelity, high processivity polymerases you can drop the extension time all together for products this short because the polymerase is so fast.

But if you are using Taq, 30 sec should be okay.

[zack]

perneseblue, on Feb 12 2009, 09:40 AM, said:

zack, on Feb 12 2009, 09:27 AM, said:

perneseblue, on Feb 12 2009, 09:25 AM, said:

you can drop the annealing time to 30sec. 1min is very long. Depending on the complexity of your template (is it genomic DNA? plasmid DNA?) can you probably go lower.

extension time is too long. Read the user manual that describes your polymerase. I would guess, dropping the extension time down to 30sec would be helpful. It can probably go lower.

yea.. this is genomic DNA... Read the user manual that describes your polymerase? u mean taq polymerase?

There are many types of polymerase on the market. With some of the high fidelity, high processivity polymerases you can drop the extension time all together for products this short because the polymerase is so fast.

But if you are using Taq, 30 sec should be okay.

what about denaturation 94- 1 min... should i change it to 30 secs too

Edited by zack, 12 February 2009 - 10:07 AM.

[perneseblue]

zack, on Feb 12 2009, 09:45 AM, said:

who about denaturation 94- 1 min... should i change it to 30 secs too

yes. Reduce that too. 30sec would be fine. But i think you can go down to 20sec.
I would also reduce the number of cycles to 25. Taq isn't a proof reading polymerase and makes a fair number of mistakes. It isn't something you usually want to use in a cloning. Furthermore, the PCR reaction does peak.

Also at around 30+ cycles, the amount of PCR product produce levels offs.. due to inhibition from pyrophosphate.

It is more efficient (time) and safer (fewer mistakes by the polymerase) to run two tubes of the PCR reaction than try to push a single tube to high yields to obtain enough product.

[zack]

perneseblue, on Feb 12 2009, 09:53 AM, said:

zack, on Feb 12 2009, 09:45 AM, said:

who about denaturation 94- 1 min... should i change it to 30 secs too

yes. Reduce that too. 30sec would be fine. But i think you can go down to 20sec.
I would also reduce the number of cycles to 25. Taq isn't a proof reading polymerase and makes a fair number of mistakes. It isn't something you usually want to use in a cloning. Furthermore, the PCR reaction does peak.

Also at around 30+ cycles, the amount of PCR product produce levels offs.. due to inhibition from pyrophosphate.

It is more efficient (time) and safer (fewer mistakes by the polymerase) to run two tubes of the PCR reaction than try to push a single tube to high yields to obtain enough product.

okey.. thank you ...i will follow ur advice .. then i will give the result soon as possible

Edited by zack, 12 February 2009 - 10:11 AM.