hello
i am trying to overlap four fragment through use of overlapping pcr:
the fragments are CMV promoter (595bp), Kanamycine resistance gene (795bp), IRES (206), and EGFP (720bp). i designed the primer with 25 bp overlap.
i amplified the different fragments using pfu polymerase, i got the bands and i extract them using gel extraction gel.
in the first overlap pcr i did as follow CMV+KAN and IRES+EGFP with equimolar and using 1 ul forward primer and 1 ul of reverse primer at 10pmol/ul, the pcr conditions as follow:
95C 3min
95C 30sec
52C 30sec
72C 30sec X5cycles
95C 30sec
55-60 30sec
72C 90sec X30cycles
72C 7min
15C hold
i got the correct size and extract them from gel.
i did the second overlapping pcr CMV-KAN+IRES-EGFP as the first one
95C 3min
95C 30sec
52C 30sec
72C 30sec X5cycles
95C 30sec
55-60 30sec
72C 2min30sec X30cycles
72C 7min
15C hold
and i got the band as in the image with correct size of 2.3kb
i did the extraction and i did the cloning of fragment usien HIPI taq to clone it inside the T-vector however i got smear and no band.
thanks for your help.
Hi Abdellaoui,
What kind of competent cell you used for cloning the final product into T-vector?
Sometimes, the endonuclease will be activated during plasmid preparation in some kinds of bacteria. You need to inhibit the endonuclease or your plasmid may be digested in the process of mini prep and lead to smear profile.
Best
hello pr. weber lin i didn't get to the T cloning because while i did the pcr using the HIPI taq and load the product in gel i got a smear. i dont know what is the possible cause of smear.
thanks for your help.
The smear probably the non-specific amplification.
Try to elevate initial temperature of annealing step and the elongation time (because your gene is about 2.3 kb) as followed:
95C 3min
95C 30sec
60~54C 30sec (decrease 2C per 5 cycles)
72C 2min30sec X5cycles
95C 30sec
52 30sec
72C 2min30sec X30cycles
72C 7min
15C hold
Good luck!
thanks a lot, i will try your advice and i will keep you informed.
Even if you are going ahead with 4 fragments your annealing temp setting should by the Tm of the fragments mentioned. It's not by the size of the fragments. Your approach with 2 fragments at a time is fine just check the Tm of those fragments once again.
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