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.

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

Gel extracting the oligos reduces the amount of shorter oligo product in your sample and thus increases the chances for a successful ligation.

Regards

Landene
Surendettement

If you are still having trouble, gel purify your oligos first on a polyacrylamide gel, then anneal the two oligos and phosphorylate them. Dephosphorylate your digested vector and gel extract that as well. Then set up your ligation in 10uL. I've done this many times and it will even work with the quick ligation kits. Good luck.


Research Papers

There is a method called Ligation assisted by nucleases (have a look at this paper in Nature protocols: https://www.nature.com/nprot/journal/v2/n9/full/nprot.2007.325.html)

Basically, you design your primers to have overhangs complementary to the restriction sites, but such that once they are ligated in, the restriction sites in the original vector are lost.

You run your ligation by mixing your circular vector + annealed primers + restriction enzyme + T4 DNA ligase in one single tube. If the vector re-circularizes, it gets cut again by the restriction enzyme. If the primers get inserted, the restriction sites are lost, and the product remains circular. The method works best with enzymes leaving longer overhangs (I have successfully used it in the past with XhoI/SbfI sites).

vector:
...CTCGAG
...GAGCTC

after cutting
...C____
...GAGCT

oligo overhang
TCGAH..... (H=not G)
____D..... (D=not C)

after ligation (restriction site lost)
...CTCGAH...
...GAGCTD...


In the past, I got >10x efficiency over self-ligation. Oligos should be non-phosphorylated, and pre-annealed (5' @95C, then slow cooling to 4C or similar).

Hi,

I read one of your query regarding annealing two primers and ligating it with a vector. I wanted to know if that worked for you. I am also interested in following similar strategy. I want to insert customized polylinker into my binary vector. Would you mind sharing your protocol. I will appreciate your help!

Ampelo!

nanaz on Fri Jan 7 17:11:44 2005 said: