very easy sticky end ligation: no colonies! - Cloning masters, please help doomed cloner! (Dec/17/2006 )
I think I should write a book about "problems that occur in cloning" . so this is very very simple straight forward ligation.
-design PCR insert with EcoRI-XbaI sites (3 bases at each end)
-Agarose purify after PCR(6 PCR reactions 50ul each ) (tried also column purification)
-Digest with REs for overnight.
-inactivate REs and ethanol precipitate.
-digest vector with EcoRI and XbaI
-Gel purify, phenol/chloroform, isopropanol precipitation (first time i dont use ethanol, wonder if it's a problem )
-Ligate 1 ul of the vector and all of the PCR insert that I have. (5 min at RT using rapid DNA ligation kit from ROCHE)
-Electroporate into very good cells (positive control is beautiful)
-Look next day to find NO colonies
So what is it now???? Please help me to figure out. ANY suggestion would be appreciated. Am I just doomed in cloning????
Note: all purification yeilds are checked by the gel.
different troubleshooting steps will be appreciated.
Some potential problems to look at:
* UV exposure of your DNA during gel extraction/imaging (do you use short wave UV?)
* Phenol in your DNA prep (do you chloroform extract to get rid of phenol?) Why do you think you need a phenol extraction at all?
* Do not gel run and extract following digestion -- no reason to if you heat kill
* No reason for using quick ligase -- regular ligase works fine for sticky ends
* Possible ligation buffer inactivity (no ATP from freeze/thaw/room temperature)
* You don't say how much ligation mix you are adding to competent cells. Less is more. Max of 5 ul into 50 ul cells.
Gel extract PCR product. This is important to get rid of PCR enzymes and dNTPs. Avoid 305 nm UV, use a column not phenol and don't bother to precipitate. Mix correct ratio of insert and uncut vector, RE buffer, REs, digest 1 hour at 37. Heat kill 20 minutes at 80C. Mix 8.7 ul of this mixture, 1 ul of T4 ligase buffer, 0.3 ul ligase at RT, wait 20 minutes. Transform 50 ul of competent cells with 2 ul of the ligation mix. A good control is to do all of this in parallel with water instead of gel extracted PCR product to test for background vector religation or lack of cutting.
Are you confident of the individual steps of your process (which I think is too complicated, also)? I'd suggest you try the following (but I'm sure you've already covered most of these checks anyway):
Once you have carried out the PCR, try doing an ammonium acetate precipitation, rather than going through gels. All you want to do is get rid of template and reagents and by-products.
Do two separate single enzyme reactions, to make sure the digestion step is working. After the reaction, heat-inactivate, then take a few ul and try ligating for 1 hr. Run a gel of this. If you don't have a band at twice the PCR product size, your enzyme is the problem.
Once you know for certain that each enzyme is behaving itself, do the double digest, heat-inactivate and test by ligation as before. This time, only do the ligation reaction for about 30 minutes, run a gel and look for a smear.
Test the vector digestion by double-digesting, EtOH precipitating, ligating (or trying to...), and transfecting your cells. You should, naturally, have no colonies.
I really, really hope this starts to work for you. Always remember, you are a sentient being, the DNA and bacteria are not! They will not beat you!
Have you considered a different set of enzymes? For myself, I have not used Xba: does it usually behave itself?
Never had XbaI misbehave on me. In my book XbaI is a good enzyme.
I agree with phage434... drop the quick ligase. Quickligase is fast but also inefficient.
kathy please give me a chance to be the co-author of ur book. because i also have a huge experience in this topic.
i just want to say did u use the right plate with right antibiotic.
once when my ampicillin stock was finished, then i make new stock and use it to make plate and culture. and i didnt find any growth and culture medium. after one week i came to conclusion that my ampicillin is not ok.
Have u tried to shorten ur digestion time
try digesting for 2 -3 hrs. Both enzymes r good that u should have complete digestion in 2 hrs.
Digest in thirty minutes to an hour.
Ligate for fifteen minutes in quick ligase.
Make sure to chill for your precipitation.
Don't use the lamp for more than 15-30 sec for visualizing and cutting out your insert.
If you don't see any bands on your gel prior to ligation, there is something wrong with your extraction protocol (that would be my guess for your problem).
Don't gel purify the vector backbone.
IME, you lose everyting and it's a waste of time.
With all due respect, I disagree with the above suggestion. The vector backbone should always be gel purified to remove any denature plasmids and any DNA fragment that was cut from the vector.
I often don't gel purify the vector and its fine. It also depends on your ligation. If you are ligating an antibiotic cassette, then your selection is so strong that it doesn't matter about background. I tend to believe that undigested vector is negligable and results in only small amount of background.
EDIT: Of course I mean plasmids that are linearised by one enzyme and do not release any insert.