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dephosphorilation of vector ends


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#1 eyes

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Posted 24 March 2011 - 08:53 AM

Hi I've digested two vectors with XhoI. Vector one has two XhoI sites and its digestion allow me to recover the insert that I should clone within vector 2, which instead includes only one XhoI site. In order to avoid recircularization of vector 2, before loading both restriction reactions on agorose for cutting the corrected bands,I treated it shrimp alkaline phosphatase (SAP). Then I load everything on agarose and cutted out the two bands (about 700 bp and 5 Kb). After that I ligated the two fragments using different molar ratios (10:1, 3:1, 1:1, 1:3 Insert:Vector) and transformed Ag1 E-Coli strain. As lower the amount of insert I used as much transformed colonies I obtained, but in all cases the colonies did non include my fragment. I loaded the ligation product on an agarose gel and what I saw were multimer of insert (that demonstrates that both ends of this fragmet are able to ligate to each other) but no evidence of circular plasmids. I don't know how can I fix this problem. Do you think I should change the molar ratios insert:vector or should I think that SAP may alter the ends of my vector?
Please help
:unsure:

#2 eyes

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Posted 24 March 2011 - 09:18 AM

sorry I forgot to tell you That I've used a fermentas SAP with the following protocol:
Plasmid 5 Kb (220 ng/ul) 20 ul
10X reaction Buffer 2,5 ul
SAP (1U/ul) 1 ul
H20 to 25 ul

I incubated 30' at 37C and then heat inactivated for 15' at 65C

#3 HBImolbiol

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Posted 24 March 2011 - 09:26 AM

I assume you are dephosphorylating your destination vector only and not the insert?

#4 eyes

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Posted 25 March 2011 - 12:04 AM

I assume you are dephosphorylating your destination vector only and not the insert?


Yes, of course




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