Help on digestion and ligation...colonies not wanted why? - (Feb/28/2006 )

I recently perform a digestion of a 4000 bp vector and a 1200 bp of insert with one restriction enzyme namely Xho1, purchased from New England Biolab. After doing so i perform ligation...and then i transfect it in a cell...

the first 2 experiment i did no cells forming on the control (which should be because they can ligate back to itself together) so i decide to increase the amount of DNA of vector and plasmid

the 3-4 experiment i see colonies but none that i wanted

then in order to stop the vector for ligating to itself i treat it with Alkaline Phosphatase, so in the end i see colonies only on the 4:5 and 4:10 but i still cant get the DNA i wanted (hence i use easyprep, then run it on the gel and digest it again xho1 and no band is showing)

then i tried to do it again only this time, in control there are 3 colonies forming, none on the 4:5 one and 4 on the 4:10 one...but again its not the one that i wanted....

i cut the vector and digest it with xho1 from the gel and its perfect....its not the restriction enzyme fault i guess, by the way the result is a sticky end product...

help me...

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my protocol:
digest the vector (for example 5 ug) with XhoI
↓check by electrophoresis (properly cut or not)

dephosphorylation of the vector (calculate how many pmole do u need for dephos.)
↓ph:chcl3 extraction
↓ppt
↓add TE

↓check by electrophoresis

then go to ligation
vector : insert= 1:10 (1 pmole:10 pmole)
and same volume of MIghty mix (ligation mix) of vector+insert

incubation in 16c for 30 min (enough) to overnight

transformation
↓colony
↓mini prep
↓check

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thanks for your reply i did all that except the incubation for 16c for 30 minutes...i did it at room temperature for 3-4 hours...yesterday i increased my ratio to 3:15...so 3ul of vector and 15 of insert...i saw colonies forming but only 4...the procedure was the same....one more thing...i use my ladder, but for some reasons all the bands wont show up...what is the reason for this? anybody?

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how much sample (insert+vector+ligation mix) do u use for transformation?
5-10 ul is better
higher amount of ligation mix reduce transformation efficiency

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the first trial i used 1vector 4 insert and i rise it up to 20ul
the 2nd one i used 1 vector 8 insert and i rise it up to 20ul

i 've also tried 2 insert 4 vector
and 2 insert 8 vector

i've tried 5 vector and 5 insert
5 vector and 10 insert

all i raised it up to 20ul

except for the last one....i used 3 insert and 15 vector and i raised it up to 30ul.

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