HELP: Ligation Problems - (Jun/14/2007 )

[attachment=3160:Initial_Digest.jpg][attachment=3159:qiagen_p...fication.jpg]I have a vector that I digested with Hind III and Spe I. I have an insert that i obtained from digesting a TOPO plasmid with HindIII and SpeI. After digestion, i run my reactions on a 0.8% agarose TAE buffered gel and I see the appropriate fragments (850bp insert and cut vector around 5.0kb). I cut the gels and purify them using a Qiagen kit with a column. I run a gel after purification and I still see my cut vector and cut inserts although the cut vector is at a great concentration compared to the insert. I calculate the ratios for ligation based on 1:3 Vector to insert (using 1bp=660g/mol) the ratios i end up using are in pmols. I usually end up with about 3ul cut vector+15ul insert in a 20-21ul ligation reaction. Problem is when i transform and plate on an Amp plate I get no growth on the cut vec (good) and no growth on the cut vec + insert (bad). I have tested my ligase and buffer and transformation technique by ligating my neighbours insert and vector (and I see lots of growth). I have practically done this for one month and I am really frustrated Now! Please any help would be appreciated. Tomorrow I am trying a new purification system cos i can only think that maybe my buffers in the Qiagen kit are somehow inhibiting ligation (possibly the PE buffer although i spin twice at that step to get rid of residual amounts). Help Me Please I have an exciting project and this is really hurting my progress!!!

PS: I have attached the gel pics:

1st Picture is restriction digestion gel (1st lane is Desired cut vector and undesired insert; 2nd lane is Undesired cut vector and desired insert)
2nd Picture is products after gel purification (1st lane is Qiagen purified "Desired cut vector"; 2nd lane is Qiagen purified "Desired Insert")


Thank you for your help in advance

To me, it looks as though you have way way too much vector. I never calculate my vector and insert ugs -- just estimate by looking at the gel. If your insert concentration is out numbered, it makes sense that you would get no growth on cut vector + insert (it is in essence simply cut vector, with the insert concentration negligible).

I'd use about 7uL insert and 0.5uL vector and ligate in 10-15uL, then transform 5uL. For controls I just set up two ligation reactions -- one with vector only and one with vector and insert. I look for minimal-no growth on vector only (some due to self ligation or incomplete digestion), and more colonies on my insert plate.

I also question whether or not you have a complete digestion with such a high concentration of vector. This would also severely reduce your ligation efficiency, and would explain the two bands you see for your vector.


Amanda

I am a little worried about the digest. It looks kind of odd to me. The fragment, although small in size is rather faint, for the backbone that intense. This is true for both the insert and vector DNA. Furthermore the high molecular weight DNA in both bands.. it looks like bacteria genomic DNA and should have been digested to bits by the restriction enzymes.

Since this experiment isn't running well, could you spare the time to do a small restiction digest.
Raw uncut vector
Vector cut with HindIII
Vector cut with SpeI
Vector cut with SpeI and HindIII

Raw uncut insert plasmid
insert plasmid cut with HindIII
insert plasmid cut with SpeI
insert plasmid cut with SpeI and HindIII

I feel somehow that your digest is not working properly. Are you sure the enzymes are digesting?

The plasmids seems to be only partially digested. Also the concentration of the insert seems to be quite low that you may need more of the insert for ligation.

it looks like the insert is being cut out. Still, it is stange that the insert band is so light compared to the plasmid's main backbone.

Hmm... just thinking, I wonder if the vector should be cut by a enzyme that cuts the plasmid multiple times, to make sure the plasmid prep is pure.

Anyway, another conventional problem is over dephosphorylation of the vector. Could overdephosphorylation be the trouble maker here?

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hi,
I actually calculated ratios based on ug vector and insert assuming 1bp = 660g/mol. My cut vector is approx. 5.2kb and cut insert is approx. 1.5kb. Based on the above gel which is loaded with 8ul of each, i estimated 75ng cut vector in 8ul and 25ng insert in 8ul. I then followed the other steps to calculate number of mols present of each. I only included the volume to give you an idea of my ligation reaction volumes. I also have used the uncut DNA control and cut DNA control. The uncut DNA on antibiotic grows, the cut DNA on antibiotic does not grow (so it is cut well). The ligation however is still waiting for kingdom come!!!
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Hi, share with your problem and also my problem now ((
I have 4.4kb cut vector and 1.1kb desired insert. I have calculated like you did for the ligation ratio 3:1 or other ratios, event I have more desired insert on the gel (10ng/ul) than you had, I did all the control like you did but I got nothing on plate. Actualy some time I got some colonies but they did not gave any result on PCR screening. My Gost, now I have only one think in my mind if the desired insert have been cut well by two REs.
Gost always test our patience (( Anyhow, if you have more idea please share with me. Good luck!

I am going to go with the above advice and do a phenol-chloroform purification of my plasmids. I am fast running out of ideas. I think perhaps my plasmids are not pure and so the ligation is being inhibited. Although i think this should not be the case since in gel purification should get rid of such things. I will also try getting more concentrated insert. Hoping for the best. Goodluck to you too.