linearisation of plasmid - pBS never gets cut :-( (Oct/07/2005 )
hi
i have been trying to cut Plasmid Bluescript with ecor1 and xho to insert cDNA. but the ligation has not been working and i even get white colonies in the negative transfomation which shows that my plasmid is self ligating. first of all eco and xho have incompatible sequences and it should never self ligate, i have also been doing the dephosphorylation to prevent all chances of self ligation.
someone please help me out!!!! 
There's no need to dephosphorylate a vector cut with two enzymes that produce incompatible ends -- in fact, I would discourage you from doing so.
Do the enzymes each cut singly? How long are you doing your double digest for, and under what conditions?
Are you gel purifying your cut vector?
How big is the distance between your XhoI and EcoRI sites? If they are too close to each other, then the second enzyme will not cut...
You can also perform the EcoRI cut and the XhoI cut separately (in different reactions) and then run them on a gel to see if your enzymes really cut.
Do you gel purify after digestion? Your original small sequence will still be in the reaction when you ligate if you don't appropriately purify after cutting.
Hope this helps.
Sounds like one of your enzymes is not cutting and hence the self-ligation. This could be for a number of reasons including as the previous poster has said that the two sites are too close or that one of the enzymes is bad or the buffer consitions wrong.
il tell you what we exactly do. we cut the vector with xho and leave the reaction for 6 hours at 37 followed by pcr purification then a digestion with ecor1 and its respective buffer.we dephosphorylate the vector with ciap and use the same for ligation.
the distance should not matter i guess coz the digestions are done at different steps.if i am wrong please let me know
thanx a lot for the suggestions
lakshmi
hi
neb web page gives information of effiency of enzymes to cut close to the end of a dna fragment. But in general cases, 1 to 3 bases are required and i suppose it is ok with your digestion.
but cip is not necesary. you can save time and DNA by cutting at the same time with the enzyme mix. (100%activity of both in neb buffer 2)
fred
1X NEBuffer 2:
50 mM NaCl
10 mM Tris-HCl
10 mM MgCl2
1 mM dithiothreitol
pH 7.9 @ 25°C
cleavage efficiency regarding DNA lenght after 2h / 20h, in percentage
EcoR I ..GGAATTCC.......>90 >90
..........CGGAATTCCG.....>90 >90
........CCGGAATTCCGG...>90 >90
Xho I...CCTCGAGG........0 0
.........CCCTCGAGGG......10 25
.......CCGCTCGAGCGG...10 75
Never forget that if you have still a little bit of supercoïled vector (bad cutting of one enzyme) you will surely trap some into your vector purification specially if you overload your preparative gel. I had this problem several times
Just to remind you 
pesji 
This should be working. How are you treating your insert?
insert is cDNA thats ligated with ecor1 adaptors,which is further digested with ecor1 that would give ecor1 and xho cohesive ends.
Just to remind you
pesji
we cant have the supercoiled dna as we do a pcr purification after the xho digestion and a gel elution after the ecor1 digestion.so i dont think that should be a problem.