I tried a ligation of 524 bp fragment into pET21 A vector
0.022 pmols of Vector-80 ng versus 0.121 pmols of Insert -50 ng
I used Ligase Weiss units 0.28 as per pet manual -On running the gel of the ligation mix after getting no transformants I saw no ligation
What changes can I make?
A bit sparse on detail. How long did you ligate for? What temperature? staggered end or blunt end ligation? PCR product or cut insert from another plasmid? gel purifiied? column purified? Dephosphorylated vector/ kinased insert?Try using fresh ATP (it is quite labile and can go off). As a check that the ligation worked, you could take an aliquot of the ligation mix and do a PCR amplifciation using a primer located in the vector (T7??) and one within your insert, or use primers spanning the insert.
Ian Teo
Ian
I did 13 hours at 16 degrees
,It was a sticky end ligation -both vector and insert cut with EcoRI,
the vector was dephosphorylated but not insert
The buffer has ATP
I have no ligation mix left to check PCR but ran it on the gel clearly showing that both vector and insert are intact
13h at 16C seems a bit excessive to me. Usually if we do a 13h ligation, it is done at 4C (o/n). Either your ligase is not working, or the ligation is being re-cut with residual EcoR1 activity. (I presume it was heat inactivated/ cleaned up before ligation. Was the insert a PCR product ? How far in from the ends was the E.coR1 site if it was? (i.e is your insert cut properly). As it is an enzyme cut insert, it should not need kinasing. As I said before ATP can degrade, so it is worth checking that (not dATP).
The kits we use (promega) contain PEG as an accelarent for ligation which is usually over in 5-30 mins at r/t. A possible problem with your extended ligation is that the ligation might well have taken place over a short period of time, but residual E.coR1 has cut the product. As ATP is labile, the rate of re-ligation may not have been sufficient to negate this. Alternatively there is some inhibitor of ligation in the mix - carried over from your insert prep??
Ian Teo
Yes EcoRI is heat inactivated and gen cleaned (in my case LMP gel was also done as large scale digestion was not complete)
Yes the insert is a PCR product -the restriction site GAATTC is 3 nucloetides away from the ends
I suspect the ligase is a problem as both Vector and insert are seen on the gel -I am plannning to do a self ligation with EcoRI cut vector to reconfirm if ligase is working along with a postiive control of Lambda Hind III
I suspect that the problem may lie in the cutting of your PCR insert. 3 nucleotides from the end of the PCR product may be a bit too close. I usually allow 4-6 bases. With such a small lead sequence in front of the PCR product, you will have difficulty in determining if it has cut. If it is a PCR product, you could try TA cloning or blunt end cloning into a vector with flanking EcoR1 sites and then cutting out with E.CoR1 Otherwise re-amplify with a new primer with longer lead sequence at the ends.The other thing that you can d, .is if your ligation efficiency is very low, after the transformation, grow in SOC media for 1h to allow expression oif antibiotic genes then transfer to 5mls of broth + anti-biotic. Grow o/n to see if there is any growth, and then plate an aliquot. Growth in media can sometimes be a bit better than on plates.
Good luck
Ian
Good luck
Ian
Dear Ian
In this ligation attempt I have done what you said as I got only 1 recombinant on plating 50 and 150 microlitres .
On growing the recombinant in SOC overnight and plating it both 5,50 and 150 microlitre, -I got a lawn-Then I diluted it and plated the same to screen for recombinants
I got the clone on replating the ligation mix -am getting it sequenced
Thanks Ian for the advice you gave My clone has been sequenced and is ready for expression It has been an enriching experience on this forum and I will keep posting queries and questions in the future too
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