4 replies to this topic

[veniesh raj]

I needed you help…


PCR condition:
This for 30 cycle....
Initial Denaturation     94 C 3 min
Denaturation              94 C 1 min
Annealing                    -       1 min
Extension                   72 C 2 min
Final extension            72 C 10 min
Hold                           16 C Forever

Annealing temperature:
Sample    Annealing temp
2 - 46 C
3 - 48.2 C
4 - 49.8 C
5 - 51.6 C
6 - 53.5 C
7 - 55.5 C
8 - 57.2 C
9 - 58.8 C

result for this annealing temperate can refer to images... ( 1 indicate maker, 2 to 9 is PCR product according to annealing temperature)



Master Mix:
Reagent                        for 1 sample
ddH2O                                15.8ul
10x PCR buffer                    2.5ul
MgCI 25mMol                      1.5ul
dNTP 10mMol                       1ul
primer F1 10mol/umol           1ul
           R1  10mol/umol         1ul
Taq polymerase                   0.2ul
DNA template                        2ul

Refer to the result, what is indicate A? This is good result or very complicate result…?What modification can I do for get better result( single band)…..?Thank for you help…!!

Edited by veniesh raj, 10 October 2009 - 12:42 AM.

[Adrian K]

Hi veniesh raj,
I have to know your expected band size before I can say anything about your pcr conditions.
I didn't see a clear marker, I suspect that you might not have enough etbr in your gel, or you stain not long enough.
I didn't see any band either, your pcr seems not working. try higher range of gradient, perhaps up to 70C
I dont see anything for A.

[veniesh raj]

adrian kohsf, on Oct 10 2009, 09:03 AM, said:

Hi veniesh raj,
I have to know your expected band size before I can say anything about your pcr conditions.
I didn't see a clear marker, I suspect that you might not have enough etbr in your gel, or you stain not long enough.
I didn't see any band either, your pcr seems not working. try higher range of gradient, perhaps up to 70C
I dont see anything for A.

Thank for you explanation.   the expected band size i don't know..where i can find this information..?? Because i am is the first person doing isolation gene from that plant. The annealing temperature suggest by the company for forward primer is 62.9 and reverse primer is 52.8.

I also want ask, how i can determine the primer i used is suitable for my plant or not??  

Edited by veniesh raj, 10 October 2009 - 11:22 AM.

[Adrian K]

Do you design the primer yourself? if you do, try to redesign the primer to get a nearer gap of annealing temperature.
If you got the reference for the primer, try to follow it. You should know where you got it from,and will probably answer all my questions.
Which gene and from which plant you isolate?
Is your template quality good enough?

Above just a few questions to be answered before we can discuss further on how to improve it.
^__^ good luck

[veniesh raj]

adrian kohsf, on Oct 11 2009, 05:57 PM, said:

Do you design the primer yourself? if you do, try to redesign the primer to get a nearer gap of annealing temperature.
If you got the reference for the primer, try to follow it. You should know where you got it from,and will probably answer all my questions.
Which gene and from which plant you isolate?
Is your template quality good enough?

Above just a few questions to be answered before we can discuss further on how to improve it.
^__^ good luck

YES, i am designs primer for my own self.. i doing research for detection or isolation monoterpene synthase gene such as myrcene and (E)-b-Ocimene from Mirabilis jalapa ( common name is four clock flower). I already search same information from journal, mirabilis jalapa plant only contain few monoterpene synthase gene such as (E)-ß-ocimene, α-farnesene, (Z)-3-hexenyl acetate and myrcene. So, i have detection or isolation of one or more monoterpene synthase gene... i can email my research proposal if you want... thank!!   This is my email address veniesh@yahoo.com. Pls email more information to me..

Edited by veniesh raj, 12 October 2009 - 11:04 AM.