To ligate the 3.5 kb DNA insert restricted with EcoRI and SalI in to a vector cut with EcoRI ,what would be better?
1. To blunt the ends of both insert and vector and ligate? or
2. To use linkers/adaptors to make SalI end of insert to EcoRI end ?
and if latter is better which is the best possible adaptor sequence? PLz help as this concern is bugging me a lot!!!! Thanx.
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Which is better? To blunt my insert and vector or use linkers?
Started by calvin*, Feb 28 2005 03:37 AM
1 reply to this topic
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calvin*
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Posted 28 February 2005 - 03:37 AM
"In my opinion, we don't devote nearly enough scientific research to finding a cure for jerks"-Calvin
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fred_33
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Posted 28 February 2005 - 03:48 AM
hi
i suppose your fragment will look like Eco -------- Sal I
AATTC----------G
G----------cagct
Hence, restriction sites are not compatible. That's why i would ligate this insert like a standard one.
but do you mean you want to change the sense of the restricion sites?
If you want to do so, you may blunt the ends and take an appropriate ligation protocol ( see topic 5292 at molecular biology) http://www.protocol-...?showtopic=5292
hope i've helped you for a few...
Fred.
i suppose your fragment will look like Eco -------- Sal I
AATTC----------G
G----------cagct
Hence, restriction sites are not compatible. That's why i would ligate this insert like a standard one.
but do you mean you want to change the sense of the restricion sites?
If you want to do so, you may blunt the ends and take an appropriate ligation protocol ( see topic 5292 at molecular biology) http://www.protocol-...?showtopic=5292
hope i've helped you for a few...
Fred.
