How to ligate ds-oligos into a vector? - (Aug/25/2004 )
I'm not quite sure about your protein isolation, but there are commercial kits (e.g. Qiagen) that have protocols for DNA Extraction out of agarose and PA gels.
mike
DNA in PAGE can by eluted into TE and 0.1%SDS overnight OR you can put it into dialysis tubing and eletroelute it.
Hi
I read your replies to enthusiast and I had similar problems and my primers also have the restriction sites at the end of them of the same enzyme to be later ligated in the sinly cut vector. I tried dephos my vector and ordered my primers 5' phosphorylated. I annealed the primers by heating 94 C fro 1 min and at room temp for 1hr. and also as suggested by some one I annealed them by placing in a water bath 70C for 1-2hr and bringing temp down slowly for a whole day say 10 each hr. I still get as many colonies on my control plate (no insert) after ligation. I even tried cutting the dephosphorylated digested vector off the gel and purify it. I did however never gel purify my insert (the annealed!! primers). How would I do that? Do I just let anneal and run on gel and purify or......???
I would be grateful if you could suggest why I am getting similar no. of colonies on my control (no insert) plate as my ligation plates?
Thank you in advance
Depending on the size of your oligos, you probably need to purify them. If they are under 30 bases, make sure to anneal properly (see below, just skip purification, add together ~800pmoles of each, anneal in 100uL, dilute as described and use in ligation). Since you are getting similar colonies on your no insert plate, your vector is not completely cut....Use more enzyme in a new reaction, and CIP for 1 hour. You can ethanol precipitate or gel extract before using in the ligation. Here are some protocols:
To purify single stranded oligos, you must run them on a 10% TBE-Polyacrylamide gel. Purify 900pmoles of primer (for ~45 bases) and mix with RNA dye (formamide based dye). Heat 2min at 90C, then load on gel. Run at 30mA until dye is halfway down. To see the oligo, you will need a fluor-coated silica plate (Ambion #10110, Fisher also sells larger sizes and amounts). Cover your silica plate with saran wrap, place your gel on it. You can view the oligos by "UV shadowing": hold the plate at a 45 degree angle above a UV Box, a purple band will be visible. The darker band near the top, if multiple bands are seen, should be your correctly sized DNA oligo. cut it out with scalpel and place in 1.5mL tube, you can break the gel piece at this point, it will help with the elution. Elute in elution buffer at RT overnight, or at 65C for 2 hours. After the elution, spin down your tube, remove the supernate and save to a new tube. Add 200uL buffer to the gel piece. Vortex and spin down, combine with the first 200uL. Ethanol precipitate with 1uL of 10% Dextran Blue to view the DNA after spinning it down. Resuspend each oligo in 10uL.
Elution Buffer: 600uL of 5M AmAc, 60uL 1M MgAc, 120uL 0.5M EDTA, 60uL 10%SDS, 5.16mL H2O.
To Anneal the purified oligos: Combine plus and minus oligos, total of 20uL. Add 10uL of Promega Buffer E and 70uL H20. Place in boiling water bath for 5min. Remove the water bath from the heat source and let cool to RT. (I use a beaker on a hot plate, and remove it from the hot plate after 5 min and leave the samples in it white it cools down.)
Take 10uL of the annealed oligos, dilute in 990uL H2O. Use 2uL of this dilution for ligation.
If you did not order your oligos with a 5' phosphate, do the following after annealing:
2uL annealed oligos, 1uL 10mM ATP, 0.5uL 10XPNK Buffer, 0.5uL H20, 1uL PNK
1hour at 37C, then heat inactivate by adding 0.5uL 0.5M EDTA, 10min 68C. Dilute to 200uL. Use 2uL of this dilution for your ligations.
This protocol was derived from Methods in Enzymology Vol 346, Designing and Characterizing Hammerhead ribozymes for Use in AAV Vector-Mediated retinal gene therapies, by Fritz et al, 2002. pp358-377.
Take 10uL of the annealed oligos, dilute in 990uL H2O. Use 2uL of this dilution for ligation.
my total primer size is 37 bp and so I guess I can skip the gel purification of the primers or can I do it using say Qiagen gel extraction kit?
also I have never used buffer E so I searched Promega and could only find buffer A, B, C and D for double digestion of DNA!!
when you say water bath do uou mean 70C
I have tried doing the technique you suggest but without the buffer E and putting the primers to anneal in a water bath bringing down temp by 10 degrees carrying on for the whole day long.
and at the end I got the colonies I said before!!
Thanks again
Hi,
I have a 25nt ds oligo insert and about 4kb 2digested vector. What rato is good to make? Can I use for the ligation, the phosphorylation reaction solution right away, or I have to clean the phosph. oligo?
Thanks
Why is it necessery to phosphorylate the annealed ds-oligo? What is the difference btw cloning a restriction digested PCR-product and an annealed ds-oligo when it comes to phosphorylation status of the ends of the insert. I have ciap-treated my vector, in one end its SnabI (blunt) and the other EcoRI. I dont get any ligation. My supervisor dont think i should try phosphorylating the linker. Can anyone explain?
Synthesized oligos are not 5'phosphorylated (unless you specifically order them that way). To prevent vector self ligation, it should be dephosphorylated (if you use two different restriction enzymes, this is not necessary). As you probably remember, either the vector or the insert needs to have 5' phosphates for sucessful ligation. So, if you dephosphorylate your vector, you must phosphorylate the oligos.
"What is the difference btw cloning a restriction digested PCR-product and an annealed ds-oligo when it comes to phosphorylation status of the ends of the insert."
Enzyme digested generated ends are still 5'phosphorylated.
Do your annealed oligos form a blunt end and an EcoRI complimentary end? Try digesting your vector with your two enzymes, gel purify (to get rid of the short vector sequence) and do not treat with CIAP. Since you have two different enzymes your vector self ligation background should be very low. Then, ligate your annealed oligos (unphosphorylated) to the vector. Your vector still has 5' phosphates so your oligos don't need them.
Thanks, I've already tried ligation without ciap-ing of the vector and it worked fine
Hi, can I ask the help from you?
you said, annealing two complementary oligos of ~40 bps DNA, how did you know the annealing works? I am working with 138 bps DNA annealing, no ligation products? hope can get some idea from you
thank you