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#1 2021-06-16 09:01:36

Nassiima
Member
Registered: 2021-06-16
Posts: 3

RF cloning first PCR

Hello everyone. I have been doing the first PCR for RF cloning for two weeks however I cannot see an expected (at 700 Kb) band instead there is a band at 200 kb. Only once I had 3 bands and one of them was the expected one. I tried it again with the same protocol and no band gained. My primers' TM are 80 and 83 C. I tried different annealing temperatures; 60, 62, 57, 54.  F primer: 54 bp and R primer: 45 bp. I would be so thankful if you give your opinion on that.

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#2 2021-06-16 12:54:05

Nassiima
Member
Registered: 2021-06-16
Posts: 3

Re: RF cloning first PCR

My project
https://www.rf-cloning.org/rf_cloning_project.php?proj_id=f156fb536c46ac5e949bb89504fef51c

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#3 2021-06-19 10:27:54

Steve Bond
Administrator
Registered: 2014-01-23
Posts: 126

Re: RF cloning first PCR

Hi Nassiima,
For the 1° reactions, you should be using an annealing temp of ~54°C. While the whole primer may register as having an annealing temperature of 80+°C, remember that the mega-primer is actually a combination of two primers. From the project page, only the green portion of the primer will bind to your insert.
I may be able to assist further if you type out your entire PCR protocol, including all reagents and the exact cycling conditions.
Best,
-Steve

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#4 2021-06-22 13:02:20

Nassiima
Member
Registered: 2021-06-16
Posts: 3

Re: RF cloning first PCR

Thank you steve. I did not genomic DNA extraction instead I picked a colony and put it in 10ul ddH2O and used 0.2ul of that as my template. I've got the expected band. I'm gonna do it another time because once I repeat it and no band was generated. 
10 X hifi Buffer 2.5 ul
dNTP 1ul
F primer  0.5ul
R primer  0.5ul
mgcl2    2ul
Betaine  6ul
Template DNA 0.2ul
hifi DNA polymerase 0.25ul
ddH2O  12.05ul
Total volume   25ul


Initial denaturation 95°c   3min
Denaturation  95°c  30s
Annealing   54°c   40s
Extention 72°c   90s
Final Extention 72°c  5min

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