LIGATION - CLONING (Mar/15/2006 )
HI, I have established my insert in a commerical pCR2.1 vector and I have PCR-ed up my product using restriction enzymes at the beginning and end of the insert, this have worked beautifully. My vector has been cut and dephosphorylated with CIAP, however when i begin to do transformations there are multiple colonies on my negative (CIAPed vector plate) and very few on my actual vector and insert plates. When i perform colony PCR using the original primers, I get positive results for my colonies and my -ve is negative and my +ve is positive, however whenever i digest the DNA after alkaline lysis there only appears to be the vector only. I was wondering whether anyone has any help or advice or any way of improving the efficiency of the CIAP, which i feel may be the problem.
Thanks
Do you gel purify your cut vector before or after CIP treatment? Maybe some of the vector is uncut which is why you have background. Having said that it would be the same for both negative control (CIP'd plasmid) and ligation plates.
It could be that only one of your restriction enzymes is cutting the vector/insert. I'm assuming you're using two enzymes, then in theory the vector shouldn't religate unless the ends are complementary. If only one enzyme cuts it will religate and the enzyme with two different sticky ends will prevent religation. Maybe digesting the vector in two steps (first with enzyme 1 and then enzyme 2, and vice verse) would help showing at least the enzymes are cutting by themselves.
Ceri
i had problems months ago with ligation.
i solved in this way: no UV and no EtBr. That is to say, gel extraction in gel witout EtBR and no use of transilluminator for the fragment.
It could be that only one of your restriction enzymes is cutting the vector/insert. I'm assuming you're using two enzymes, then in theory the vector shouldn't religate unless the ends are complementary. If only one enzyme cuts it will religate and the enzyme with two different sticky ends will prevent religation. Maybe digesting the vector in two steps (first with enzyme 1 and then enzyme 2, and vice verse) would help showing at least the enzymes are cutting by themselves.
Ceri
I have cut my vector with PST1 only, CIAPed and then gel extracted. I am almost certain that my enzymes are cutting the vector and that they are working properly because i have run undigested alongside the digested vector and also by using the enzyme to something i know it does work in. However my main problem once i have gel extracted is the lack of conc of the DNA, which is berely noticeable on a gel. I have used many commercial kits to try and help this problem i.e. Mini Elute kits, SureClean kits and nothing seems to work. Thanks
Do you gel purify your cut vector before or after CIP treatment? Maybe some of the vector is uncut which is why you have background. Having said that it would be the same for both negative control (CIP'd plasmid) and ligation plates.
It could be that only one of your restriction enzymes is cutting the vector/insert. I'm assuming you're using two enzymes, then in theory the vector shouldn't religate unless the ends are complementary. If only one enzyme cuts it will religate and the enzyme with two different sticky ends will prevent religation. Maybe digesting the vector in two steps (first with enzyme 1 and then enzyme 2, and vice verse) would help showing at least the enzymes are cutting by themselves.
Ceri
I have cut my vector with PST1 only, CIAPed and then gel extracted. I am almost certain that my enzymes are cutting the vector and that they are working properly because i have run undigested alongside the digested vector and also by using the enzyme to something i know it does work in. However my main problem once i have gel extracted is the lack of conc of the DNA, which is berely noticeable on a gel. I have used many commercial kits to try and help this problem i.e. Mini Elute kits, SureClean kits and nothing seems to work. Thanks
You can try to insert during the gelextraction an additional drying step (10 min @ 45 °C) to evaporate all ethanol from the column. Also try to heat the water, you are eluting with, to 45 °C.
I always add less water in the eltion step than described to concentrate the DNA.
Rocher
PCR initiated from transformation products can give rise to false positives if the primers anneal to the insert sequence due to excess DNA insert from the ligation reaction that is spread as part of the transformation reaction onto bacterial plates. Typical ligation protocols utilize 5 pmol of insert PCR product in a 20 µL reaction (0.25 pmol/µL) with 0.5 pmols of vector. Assuming that all the vectors acquire a single copy of insert, 4.5 pmols (0.225 pmol/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09 pmol) is electroporated and diluted 1:9 with SOC media (0.01 pmol/µL). If 100 µL (1.0 pmol) is spread on a 55.4 cm2 plate (0.018 pmol/mm2) and a 4 mm2 agar pick is used, then 0.072 pmols (4.35 e 10 molecules) of background insert are available for a 100 µL colony PCR assay.
To avoid this problem, at least one primer should anneal to the vector. Use of the Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification.
Excess CIAP allows contaminating exonucleases to nibble the ends of the vector, rendering it unable to contain an insert. Regardless, dephosphorylation reactions never go to 100%. But the fact that you have fewer colonies (rather than > 2x colonies plus insert) usually indicates a ligation failure. I recommend switching to SAP which can be used simultaneously with the restriction enzyme you use to cut the vector if it cuts at or below 37C.
It is possible that the restriction sites on your primers are too close to the ends of the DNAA fragment for efficient cutting. See the NEB and Fermentas catalogs for information on the minimum distances required.