Primer design and need help - (Feb/12/2009 )
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.
What bands r these? PCR products ???
Hi,
Your pcr condition is not optimized. PCR normally will produce a huge amount of product, regardless of the specificity.
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.
could it be genomic DNA (plus intron) ?
please give more details
little mouse on Feb 12 2009, 07:42 AM said:
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
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.
perneseblue on Feb 12 2009, 09:25 AM said:
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?
zack on Feb 12 2009, 09:27 AM said:
perneseblue on Feb 12 2009, 09:25 AM said:
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.
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:
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