ligation help - (Sep/17/2004 )
QUOTE (beccaf22 @ Jan 29 2006, 08:25 PM)
Maybe there is some nuclease in your purification reactions... especially if you can see the purified DNA right after purification but not 24h later from the same sample??? ligate and transform quickly??? clean up your gel box and use new razors etc???
hth
hth
I did both. there is no difference.
-jpsi-
the only other things I can think of now are to switch to all new reagents, use a positive control (insert + vector) to test ligase and a positive control (pure circular plasmid-go ahead and add ligase and ligation buffer) to check for nuclease contamination...
In addition, your final concentration should be at least 10-20ng/ul in the ligation 1-2 ng/ul favors recircularization... take into account the size of the vector/insert when determining ratios according to promega use from 3:1 to 8:1 insert:vector ratios...
Hope that helps
-beccaf22-
QUOTE (beccaf22 @ Jan 30 2006, 02:39 PM)
the only other things I can think of now are to switch to all new reagents, use a positive control (insert + vector) to test ligase and a positive control (pure circular plasmid-go ahead and add ligase and ligation buffer) to check for nuclease contamination...
In addition, your final concentration should be at least 10-20ng/ul in the ligation 1-2 ng/ul favors recircularization... take into account the size of the vector/insert when determining ratios according to promega use from 3:1 to 8:1 insert:vector ratios...
Hope that helps
In addition, your final concentration should be at least 10-20ng/ul in the ligation 1-2 ng/ul favors recircularization... take into account the size of the vector/insert when determining ratios according to promega use from 3:1 to 8:1 insert:vector ratios...
Hope that helps
Yeah. I will check for nuclease activity. But I simply bought new buffer and ligase.
3-90 pmol of ends... it is much less than 10-20 ng of plasmid per microliter, and it is in company's protocol. Why 1-2 ng favours recircularization? Do you have some reference? Sounds like interesting issue
. -jpsi-
QUOTE (jpsi @ Jan 30 2006, 03:44 PM)
Yeah. I will check for nuclease activity. But I simply bought new buffer and ligase.
3-90 pmol of ends... it is much less than 10-20 ng of plasmid per microliter, and it is in company's protocol. Why 1-2 ng favours recircularization? Do you have some reference? Sounds like interesting issue.
3-90 pmol of ends... it is much less than 10-20 ng of plasmid per microliter, and it is in company's protocol. Why 1-2 ng favours recircularization? Do you have some reference? Sounds like interesting issue
.The only reference i have is my old PI... I was doing some digests to remove an insert ie: cut with XhoI then religate so that the 600bp insert XhoI to XhoI was removed (doing 5' end deletion analysis of my promoter) he suggested using 1-2ng/ul to promote recircularization above re-inclusion of the insert--it wolrked and I avoided having to purify... He used the same numbers for avoiding concatomerization when both sticky ends are the same in ligations... so we ligated for 0.5-1hr at 10-20ng/ul then diluted to 1-2ng/ul and let go overnight to prevent concatomers....
Just his instruction in this case, that is the only reference i have... usually I have to know why, but at the time it made sense to me, so I didn't question this time... and it worked so I never went back...
When I say 10-20ng/ul I mean of both plasmid and insert so total final concentration of both is that what you meant also?...
I also agree that 10ng/ul may be alot, particularly after reviewing the pGEM T-easy protocol... maybe there was some other reason we used such a high concentration that time... I don't know now I am confused it looks like t-easy uses somewhere between 5-10ng/ul total insert+vector concentration...
hope that helps...
-beccaf22-