problems in ligation/transformation - (May/24/2007 )

Hi everyone,

I have been doing ligation & transformation the last 3 weeks but nothing has been working! I am sure of the antibiotics added to the plate, miniprep, & the enzyme used for digestion. Everything works quite fine till the ligation step. I usually incubate overnight the ligation mixes at room temperature and then transform the plamid into DH5-alpha cells and incubate them overnight at 37c. However, I do not get any colonies!! I have tried both commercial & electrocompetent cells yet no colonies. I do not see where the problem is especially that it used to work before.

I need your help on this asap.
I look forward to hearing your reply.
Thank you!

Baraa

Baraa,

We need more info from you. What's the insert size? Which plasmid are you using? Are you using a transformation control?

Mous

Hi

The plasmid I am using is pRS425 (~6kb). As for the insert, it is around 1kb.
Yes, I use a transformation control yet no colonies are growing on both the control and sample plates.

Would the size of the insert affect the presence/absence of colonies? I mean let's suppose the insert does not ligate with the plasmid. Since ligase is added, would not the plasmid close on itself (ignoring the insert)? And if it would, should not it then grow on the plate?

Let me know if you need more info.
Thank you.

Baraa

No colonies on your control plates means that your competent cells are not competent! if you're using commercial cells call the company, if you're doing it yourself re-visit your protocol. One more thing, on which RE site(s) are you ligating your insert?

Please answer so I can help you.

Mous

You could be overdigesting your vector and insert and this could prevent them from ligation as well. What enzymes are you using?

Have you verified the DNA conc on gel after purifying from gel?

I do not think overdigestion is the problem. I always run the digested vector and insert on agarose gel to do gel extraction.
The size of the bands are fine and they correspond to the right size of the plasmid/insert. The enzyme I have been using lately is SacI.

I have repeated the whole process again and again, starting with growing the bacterial strain in (LB + ampicillin) medium overnight, digestion, gel extraction, ligation and transformation. Before setting up the ligation mixes, I run a small sample of vector and insert on agarose gel to determine how much I should add of each.

I do not do the competent cells but I am sure it is not the problem either. In fact, my friend did transformation (using same cells) last week and it worked quite fine. Besides, I used to get it working too but now I don't!! I do not understand why.

Today, I have transfomed different samples of vector and insert (varying the concentration of the insert). I am waiting for the results. But in the meantime, I can not think of any solution.

Thank you!
Baraa

One cannot judge by looking at the gel picture if the DNA is over digested or not. Even if the enzyme chews up an extra base, you will not get any ligation and no clones.

How long do you digest the DNA? Is Sac I the only enzyme you are using?

Has anyone else in the lab got cloning with the ligase you are using? Ligase and the buffer might not work if they were handled improperly.

Good Luck !!!

yes, more info.

I would like to know if the ligation strategy you are using. What is the vector and insert cut with?

Do you dephophorylate your vector? If so what are the conditions. Overdephosphorylation is bad.

What is your vector to insert ratio? How many ng of insert to how many ng of vector

Can you confirm that your ligase and ligase buffer are working? Has anybody else in the lab conducted a sucessful ligation recently. The ligase enzyme in particular goes bad rather easily. You can confirm ligation by running some of the ligation mix (post ligation) onto a small narrow well gel. You should be able to see high molecular weight bands if ligation was successful.

When you repeat, do you use the same insert and vector DNA? Or have you been making it fresh each time. Which component has been in common for all 3 failed ligations. It is useful info to know as it can help pinpoint the bug.

Hello all molecular biologist,

I have a big problem in order to find a solution to purify my cDNA synthesis...

I use a Chroma Spin Columns (Clontech; TE-400), but the results are too bad ! These columns are spin columns packed with gel filtration resin to rapidly purify and size select nucleic acid samples. The columns can be used to purify single- or double stranded DNA or RNA from contaminants such as salts, solvents, enzymes, or proteins. Molecules larger than the particular matrix pore size are eluted out of the column while smaller molecules are retained inside the column matrix. As a result, these columns are ideal for size fractionation of libraries or for removal of primers.

I would like to recover the fragments higher than 400 bp and I don’t find this on the web… (in another supplier that Clontech)

If you have a idea, please help me...


Thanks for informations

One of the ways in which this is sometimes done is to run a gel. After the gel is run, the short fragments are cut off the gel, and the current direction reversed. The gel is run backwards to collect the now size-selected DNA fragments in the well. Try this first with a marker lane to debug your technique. You can also use S-500 or S-1000 sephacryl in microspin columns (such as from Biorad or Bio 101) or flowthough. See, for example, http://www.cshprotocols.org/cgi/content/fu.../2/pdb.prot3917
This is likely what is in your chroma spin column.