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Sorry if this is a repeat topic, I have looked/searched.
I have a vector that I am trying to give dual resistance to depending on cell line I want to express in.
A coworker said I could just put the puromycin cassette into the MCS but I am just wondering if this will cause problems? Two genes in one multiple cloning site?
It's a C1 vector but if I put a stop codon after the first gene, will the second one still express?
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Primer Check?
17 July 2012 - 02:02 PM
Hi! I will try to keep this as short as possible:
I am trying to clone out the eGFP from this vector http://www.synthesis...or/pEGFP-N3.pdf and replacing it with the mCerulean (sequence here: http://www.einstein....revFinished.pdf) that I PCR out from a C1 vector.
So because it is an N3 vector, and the fluorophore is on the back, these primers would be appropriate to clone out the mCerulean:
Forward: GATC ggatcc A ATG GTG AGC AAG GGC GAG (Added the one A bp to put in frame from N3 to N1 position)
Reverse: GATC tctaga TTA CTT GTA CAG CTC GTC CAT (Added nothing)
Or, should I add two addition base pairs to the forward primer because it is coming out of a C1.
Any help is extremely appreciated!!
Thank you
I am trying to clone out the eGFP from this vector http://www.synthesis...or/pEGFP-N3.pdf and replacing it with the mCerulean (sequence here: http://www.einstein....revFinished.pdf) that I PCR out from a C1 vector.
So because it is an N3 vector, and the fluorophore is on the back, these primers would be appropriate to clone out the mCerulean:
Forward: GATC ggatcc A ATG GTG AGC AAG GGC GAG (Added the one A bp to put in frame from N3 to N1 position)
Reverse: GATC tctaga TTA CTT GTA CAG CTC GTC CAT (Added nothing)
Or, should I add two addition base pairs to the forward primer because it is coming out of a C1.
Any help is extremely appreciated!!
Thank you
