Blunt end ligation help - (May/25/2006 )
Hi all,
I have a double stranded 26-mer (which I made by annealing two complimentary oligos) which I want to clone in a blunt ended linearized M13 vector (I did a restriction digest with Eco RI and then removed the 5' overhangs/stickyends by Mung Bean Nuclease). I am not sure how to do it. I looked in the Bible (i.e. Sambrook et. al) and it said to phosphorylate the insert, dephosphorlate the insert and then use T4 DNA ligase. However it said to keep as little volume as possible ( under 20 micro liter). I cannot do that since after annealing and phosphorylationg the insert, the volume is 150 micro liter and I cannot purify it because I have too little DNA in it. So what should I do. If anybody can come with a few numbers for ligation reaction, it would be great.
hi
use butanol to purify your sample. Butanol works by dehydrating. So if you have 150 µl, add 500µl butanol, vortex Strong for 15', add an other microliter of butanol (or fill the 1,5ml tube with butanol) homogeneize it by inverting several times. spin 10' full speed.
2 possibilities.
- you see only one phase. Pipett it carefully and wash by 70%ethanol / dry / resuspend in appropriate volume.
- you see two phases. Pippett the upper one careffully (Butanol partially hydrated, it's like pipetting chloroform) and redo the butanol addition/spin
for dephopshorylation, promega protocols recommends to do a first dephospho at 37°, and to add the required amount of phosphatase and to do the second dephosphorylation at 56°
use butanol to purify your sample. Butanol works by dehydrating. So if you have 150 µl, add 500µl butanol, vortex Strong for 15', add an other microliter of butanol (or fill the 1,5ml tube with butanol) homogeneize it by inverting several times. spin 10' full speed.
2 possibilities.
- you see only one phase. Pipett it carefully and wash by 70%ethanol / dry / resuspend in appropriate volume.
- you see two phases. Pippett the upper one careffully (Butanol partially hydrated, it's like pipetting chloroform) and redo the butanol addition/spin
for dephopshorylation, promega protocols recommends to do a first dephospho at 37°, and to add the required amount of phosphatase and to do the second dephosphorylation at 56°
The oligo (26 mer) that I am looking to purify is only about 180 femato moles. Even though I peform a butanol extraction, I don't think I will see the DNA precipitant.. Is it okay to use the phosphorylation mix for ligation instead.. Phosphorylation mix contains phosphorylated and annealed oligos. Plus debris of inactivated kinase and buffer. Should that hurt the ligation in any way (I am planning to dephosphorylate the vector)
Kush