very easy sticky end ligation: no colonies! - Cloning masters, please help doomed cloner! (Dec/17/2006 )

thank you so much for yoru help!

i attached the picture of the ligated plasmid:
1: marker 600ng
2: unligated plasmid (0.5ul)
3: ligated plasmids (1ul each) in 1x (correct) and 5x (wrong) DNA dilution buffer. I did this to check if high concentration of dilution buffer that i was using by mistake before interfered with my ligations. It seemed to ligate fine too however.

Thank you for the method of cutting DNA under UV. I have one question: how much of DNA do you load on the gel? this time i only loaded 1ug... I dont think with a band thin like that you can sacrifice some for visualization.

About competant cells, i ran positive control two experiments ago and it seemed fine.

I dont have anyone to ask, that is why i am so desperate. actually noone is involved in cloning in the lab here except me and the only girl who was involved some time ago is curretnly writting her phD thesis and submitting in two days so she is SUPER busy to look at my clonings.

THanks for the photo. THe DNA looks ligated.

Emm... I ususally cut 15ug to 25ug.... it is alot and many (including my supervisor) considers this wasteful. But if it gets me where I want with plenty of DNA to burn, I am happy.

Urm... yup 1ug is a little small, to sacrifice DNA.

If your real experiment consist of linearising your vector with EcoRI and XhoI. No significant insert being excise, you could try skipping the UV step. Just column purify or phenol chloroform after the digest.

Alternatively bring a timer into the translumination room/dark room. And expose your gel to UV for no more then 1min. Best to keep it close to 30sec.
First make sure the transluminator is set at low power/or long wavelenght.
I used to have my blade out and ready before even turning on the uv. Under UV, I also used to make only the cuts in the gel slab first, 4 cuts. Turn off the UV. Turn on the normal light and only then recover the gel slab

thank you for your suggestion! my professor has also suggested i skip the gel purification all together and just ethanol precipiate after the RE digestion. so i will try to do that first only with the positive control. i am just trying to religate the vector on itself anyway, i:ll post tomorrow if i have any colonies. keep your fingers crossed for me please!!!!!!!

for those who are still following: i guess my thread looks like a mexican serial movie now

like my PI suggested i digested 10ug of the plasmid with EcoRI (there is some insert inserted only in the EcoRI site), inactivate EcoRI, ethanol precipitate, and add ligation buffer and ligase. electroporate together with a positive control.

result: positive control has more than fifty healthy looking wonderful colonies. my plasmid has ONE. which i am not even thinking of checking.

so i have done no UV, no gel extraction, no phenol purification. my plates are fine, e-coli is fine.
so can not I firmly say already that this stupid ligation mix is terrible while electroporated!!!! the hand out that comes with it clearly says it should be purified BEFORE electroporating (we dont have those columns, and ethanol precipitation didnt work). what do you say?

umm.. your plasmid is cursed! Evil radiates from it!


Can you use a different plasmid prep for the cloning?
Assuming you must use this plasmid prep;

I invoke the baseless assumption that the raw plasmid prep is dirty and somehow inviting 'evil' into your cloning.

I would try phenol/chloforming the raw plasmid, 2 or 3 times. If you have pronase in hand, could use that prior to the phenol/chloroform.

If there isn't enough raw DNA (as phenol/chloroform results in the lost of some DNA) , I might consider retransforming the plasmid back into cells and do a midiprep to extract more plasmid DNA.

thank you again! Happy new year!

actually i also thought so and prepared new prep (old did look dirty on the digest) and now i am trying it with two types of ligation mixes. lets see what happens

It worked!!!!!!!!!!!!! i have changed the ligation system and there are beautiful colonies on the plate! have to check them of course but at least there is something to be checked. thank you so much!!!!

For sticky end ligation (even self ligation) I had passed more than one month and tried everything. Now atlast I got it.
Here are some points which I did.
Make new mini prep and extracted using the Qiagen column.
Changed the ligation buffer (home made)...
==
I recommend BRL's ligation buffer (which contains PEG) for blunt end ligations. 5x Blunt End Ligation Buffer (BRL Technical Bulletin 5224-1 1992.): consists of 250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000. To prepare 10 mL of buffer, weigh 2.5 g of PEG 8000 in a 15 mL Falcon 2097 tube that has been treated with antistatic spray or wiped with a sheet of fabric softener. Microwave a 100 mL bottle of Type I water for 1 minute on High. Add 4.4 mL of hot water and 2.5 mL of room temperature 1 M Tris-HCl, pH 7.6 to the PEG and immediately mix to dissolve. Cool the mix to room temperature and add 500 µL of 1 M MgCl2, 500 µL of 100 mM ATP and 50 µL of 1 M DTT to a final volume of 10 mL. Aliquot for storage at -20°C. (If you do not use hot water, the PEG will require an overnight dissolving step.)
==
It had done wonders for me.
Hope it will help you too.