3 replies to this topic

[vinee2]

Hi frnds

I am new to cloning ...and I have a question to ask...I am cloning a gene inside a low copy number plasmid...and in the MCS the two restriction enzymes sites have only 6bp difference hence are close by....I performed a double digestion of this plasmid and insert and got colonies after ligation...with 2-3 colonies on vector control plate...I ran the plasmid prepared from these colonies to compare that of the untransformed plasmid..and instead of the transformed plasmid running behind in the agarose gel than the untransformed..i observed that the transformed plasmid ran a little ahead of the untransformed plasmid....on restriction digestion no insert was obtained but plasmid was seen on the gel....I have two questions
1. Is one of the enzymes not working and so religation of vector is taking place cos colonies are there after transformation..
2. If two restriction enzymes sites are present at 6 bp difference is there a possibility that double digestion will not work? do i have to redesign the primers??
3. Is alkaline phosphatase treatment necesssary for ligation in this case...

Thanks in advance...any help will be appreciated

Edited by vinee2, 01 March 2012 - 08:27 AM.

[zogene]

Dear vinee2

Regarding to your questions:
1- Not necessary I often have clones in my digested vector actually you can not guarantee digestion 100% if you want to make sure about your enzymes just digest your vector with ones alone then run it on a gel you can easily tell if it digests or not or transform your linearized vector and compare it with undigested one.
2- There are also 6 bp between mi restriction sites and I had no problem with that at all, so it shoild be fine with you either.
3- It depends if your enzymes have compatible ends or not check at NEB website, some people do it any way.

I hope this is help you and good luck.
reagrds,
zogene

Edited by zogene, 01 March 2012 - 11:44 AM.

[vinee2]

thanks zogene....i am using BamH1 and EcoR1 and doing a double digestion with buffer 3 and also add BSA to the reaction...and both cut sites are only at a difference of 6bp....so i was thinking if instead of double digestion would a sequential digestion help...and also which enzyme should i use first between EcoRI and BamH1

[zogene]

Dear vinee2

First I will recommend EcoRI buffer instead of buffer 3 and the cut sites are fine 6 bp are enough besides it does not matter which is first, if you want you can do seq digestion but both if your enzymes work fine in the buffer so no need for it but do not incubate for more than 1 hour.
good luck