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ligating small inert into a large vector - (Jan/12/2011 )

I am trying to ligate an insert of around 125bp into a vector of 11kb in length. I am using Kpn1 and EcoR1 sites both in insert and vector. I have double digested both insert and vector using the best buffer for double digestion as suggested by Roche. I have also adopted gel purification after digestion. Finally after transformation I do not get any colonies. I am confident about transformation part because I have used the best competent cells and the method I follow there is totally fine for me otherwise in case of other inserts that I have already worked with (but ofcourse the restriction sites were different there). I would really appreciate some insights into this problem.

-laborat-

Do you have any controls if the double restriction was OK? Did you see the cleaved products? In my experience a low transformation yield is usually due to poor restriction. I would not suspect EcoRI because (at least in my hands) it is very robust, but for KpnI I honestly don't know. How did you generate the 125 bp product? Did you leave enough space for the restrictase to cleave?

-BioMiha-

BioMiha on Thu Jan 13 11:27:43 2011 said:


Do you have any controls if the double restriction was OK? Did you see the cleaved products? In my experience a low transformation yield is usually due to poor restriction. I would not suspect EcoRI because (at least in my hands) it is very robust, but for KpnI I honestly don't know. How did you generate the 125 bp product? Did you leave enough space for the restrictase to cleave?


I did not use any controls to check double restriction digestion but I have checked the efficiency of restrictions enzymes individually on the same plasmid. Digestion efficiency seems to be fine. The 125 bp product was generated by annealing two long primers of 70 bp and extension of their overhang ends at the 3' (Pardon me for i said PCR before, infact this was just extension of primers). I have checked this product on the gel for its size. This 125 bp product possesses those restriction sites at its ends and I have made sure there is enough space (4 bases before the restriction sites) for the restrictase to cleave.

-laborat-

just to double check. Do the inserts overhangs complement the overhangs on the vector? KpnI leaves a 3' overhang while EcoRI leaves a 5' overhang

-perneseblue-

perneseblue on Fri Jan 14 04:57:46 2011 said:


just to double check. Do the inserts overhangs complement the overhangs on the vector? KpnI leaves a 3' overhang while EcoRI leaves a 5' overhang



the vector is also digested by the same enzymes. and the overhangs complement

-laborat-