3 way Ligation Issues... - (Aug/29/2008 )
I have been attempting a 3 way ligation for the past month and 1/2 but haven't had any success. I started by PCRing my 2 inserts with primers adding the SacI and AscI site to fragment A and an AscI site and BglII site to B (thus, when I cut my vector with Sac and BglII the two inserts would only be able to ligate together and into the vector). After PCR purification, I cut fragment A with SacI and AscI (1 digestion, 37C for 1 hour followed by 20 minutes at 65C), B with AscI and BglII (same as A but 2 digestions starting with AscI), and my vector with SacI and BglII (2 digestions, starting with Sac).
After gel purification I've set up several ligation reactions with varying ratios of inserts:vector. I've tried 1:1:1, 2:2:1, 3:3:1 (A:B:vector). I've used anywhere from 100ng-400ng of DNA in each reaction. The ligase and buffer should be fine, it's new and I've used 2 different tubes. I've made plates and Amp a few times (but used same concentration, 100ug/mL). Colonies are growing but when I digest the minipreps all I've gotten is empty vector. I've recut the vector thinking I was only getting a partial digestion (which still may be true but there's no way to tell if I've gotten full or partial digestion, is there?) but still nothing.
I've also tried ligating them into TOPO vectors but I wasn't getting any colonies with TOP10 cells or dH5a cells after multiple attempts. Any suggestions? Any problems with my protocol? Many thanks!
Sara
hmm....
how many base pairs were add to skirt the AscI, SacI and BglII restriction site of insert A and insert B.
Are the SacI and BglII sites on the vector close?
Might there be problems from overexposure to UV?
How much DNA are you cutting? Is 1hour enough time for the amount being digested? Could this be a digestion problem?
Did you dephosphorylate the vector? Is so how much, in what volume, how long and with how many units of enzyme.
Could you run a gel on your ligated DNA? This is to see if the DNA is actually ligating. Even though the T4 ligase you are using is new, it is best to see if reaction is actually going.
That being said, the recovery of vector molecules is odd. How many colonies are you recovering? 10s? 100s? 1000s?
Did you check that the vector was linearised with SacI before digestion with BglII (since SacI digestion first). Was the vector linearised?
Did you do a vector only control? How many colonies were recovered? How did that compare to the what the vector+insert ligation.
How are you recovering your cell, in SOC? And how big is are the inserts? I am just wondering if the inserts are very big and thus the high recovery rate of empty vectors.
PS: You don't need to heat kill your inserts, if you are gel purifying. The Amp is a little on the high side, you can drop it down to 50 or 25 ug/ml to safe cost.
how many base pairs were add to skirt the AscI, SacI and BglII restriction site of insert A and insert B.
Are the SacI and BglII sites on the vector close?
Might there be problems from overexposure to UV?
How much DNA are you cutting? Is 1hour enough time for the amount being digested? Could this be a digestion problem?
Did you dephosphorylate the vector? Is so how much, in what volume, how long and with how many units of enzyme.
Could you run a gel on your ligated DNA? This is to see if the DNA is actually ligating. Even though the T4 ligase you are using is new, it is best to see if reaction is actually going.
That being said, the recovery of vector molecules is odd. How many colonies are you recovering? 10s? 100s? 1000s?
Did you check that the vector was linearised with SacI before digestion with BglII (since SacI digestion first). Was the vector linearised?
Did you do a vector only control? How many colonies were recovered? How did that compare to the what the vector+insert ligation.
How are you recovering your cell, in SOC? And how big is are the inserts? I am just wondering if the inserts are very big and thus the high recovery rate of empty vectors.
PS: You don't need to heat kill your inserts, if you are gel purifying. The Amp is a little on the high side, you can drop it down to 50 or 25 ug/ml to safe cost.
Our primers were 32-38 bp in length, with 30 bp binding to the sequence.
The Sac and Bgl sites on the vector are close (which is why we cannot see if the vector is completely cut). I just found some plasmids that were ligated into the Sac and Bgl sites so I will digest those so I can see if I have complete cutting. I have not tried running a gel after the Sac digestion but will try that as well.
I don't think UV overexposure is the problem. I try to cut as quickly as possible and keep it under 10 seconds.
The number of colonies varies from 10 to over 200 (I usually plate different amounts of bacteria). I am recovering with SOC and my inserts are 3K and 3.5K. Today was the first day I did not have colonies on my control (vector-only) so hopefully I successfully ligated in one fragment but we'll after mini prep tomorrow... Thanks!
umm... not exactly the answers I am looking for.
Restriction sites need to be flanked on either end by a few basepairs before it can be cut by the restriction enzyme. As a rule of the thumb it is 6bp on either end. Although a number of enzyme can do with less and a few (eg NotI, NdeI need more, 8bp)
eg
BamHI site : GGATCC
Binding sequence : ATGATACGATCCCTA
Guard : CGATCA
Primer :
CGATCA GGATCC ATGATACGATCCCTA
This also means that restriction site in a vector too close together will not cut. I am not sure if any of this info in applicable to your situation. I need more info about the primers your are using. And your digestion conditions.
However since you are getting colonies, things might well be working now.
So best of luck. I hope you get your clone 
.
After gel purification I've set up several ligation reactions with varying ratios of inserts:vector. I've tried 1:1:1, 2:2:1, 3:3:1 (A:B:vector). I've used anywhere from 100ng-400ng of DNA in each reaction. The ligase and buffer should be fine, it's new and I've used 2 different tubes. I've made plates and Amp a few times (but used same concentration, 100ug/mL). Colonies are growing but when I digest the minipreps all I've gotten is empty vector. I've recut the vector thinking I was only getting a partial digestion (which still may be true but there's no way to tell if I've gotten full or partial digestion, is there?) but still nothing.
I've also tried ligating them into TOPO vectors but I wasn't getting any colonies with TOP10 cells or dH5a cells after multiple attempts. Any suggestions? Any problems with my protocol? Many thanks!
Sara
Bad news, I fear. According to the NEB website, Sac I and PCR don't mix. You might have to think up another RE.
Restriction sites need to be flanked on either end by a few basepairs before it can be cut by the restriction enzyme. As a rule of the thumb it is 6bp on either end. Although a number of enzyme can do with less and a few (eg NotI, NdeI need more, 8bp)
eg
BamHI site : GGATCC
Binding sequence : ATGATACGATCCCTA
Guard : CGATCA
Primer :
CGATCA GGATCC ATGATACGATCCCTA
This also means that restriction site in a vector too close together will not cut. I am not sure if any of this info in applicable to your situation. I need more info about the primers your are using. And your digestion conditions.
However since you are getting colonies, things might well be working now.
So best of luck. I hope you get your clone
.My primer sequences for each end are: 6 bp-RE site-20bp. 6 bp SacI 20 bp and 20bp BglII 6bp
For the middle primers they have 15 bp on both sides of the RE site. 15 bp AscI 15bp
For the vector my digestion reaction is as follows:
1X buffer (NEB 4)
100 ug/mL BSA
40 units of SacI
Amount of DNA varies, with vector I've been using 2ug-5ug.
Water to 50 uL
1 hour-75 minutes at 37C.
Add:
1X buffer (NEB3)
100 ug/mL BSA
40 units of BglII
Water to 100 uL
1 hour at 37C followed by 20 minutes at 65C.
I'm digesting the minipreps now, so hopefully it's ligated in...
Could you put a link to this site? Thanks!
Could you put a link to this site? Thanks!
First of all, the website is http://www.neb.com/nebecomm/tech_reference...ion_enzymes.asp?
More importantly here, though, is ignore what I just said about Sac I and PCR. My brain is fried right now
. The problems are with Sal I, not Sac I... 
Having said that, Sac I doesn't like high pH or high salt.
so the primers all have a 6bp guard (at least). I guess the problems isn't there. The digest looks fine too....
How did the current ligation go?
How did the current ligation go?
No dice on the ligation... Still getting vector religation. Funny thing is we had no colonies on the no insert control, 10 colonies on the 1:3 vector:insert plate, and 25 colonies on the 1:10 vector:insert plate so I got my hopes up.
Might dephosphorylating the vector help? I'm pretty green so I haven't heard of this...