6 replies to this topic

[Orangistae]

I'm trying to clone some genes into a vector by amplifying them and ligating the PCR products into a SmaI(=blunt-ended cutter)-digested plasmid, but I've been having trouble with it and I've narrowed the problem down to the digestion part. When I run some of the digest on a gel it looks fine, with a single bright band, in contrast to the double band of the undigested vector, but if I transform the digested, unligated vector into E. coli I get heaps of transformant colonies, which seems to indicate that there is a very high background of uncut vector. Can anyone help me work out what the problem is and what I can do about it?

I've tried the digest two times so far, using the same SmaI (Promega, and nowhere near its expiry date), buffer, and DNA sample [which was recently purified and is in EB buffer (i.e. 10mM Tris, pH 8.5) with 1mM EDTA]. The first time, I used 6ul DNA with 2ul each of enzyme and buffer in a 20ul reaction mix and incubated at 25 degrees (SmaI's optimal temp) overnight, before heat-inactivating, treating with TSAP, and transforming 0.125ul of the mixture into E. coli. The second time, I decreased the amount of DNA in the digest to 2ul, incubated the digest for 4hrs, and used 0.25ul for transformation (didn't bother with the TSAP). I got lots (>50) of colonies both times, although somewhat fewer the second time due to the smaller amount of DNA, and when I do the actual ligation and try the transformation with that, I don't get any more colonies compared to using the equivalent amount of 'digested' vector.

Any help or advice would be much appreciated.

Edited by Orangistae, 23 April 2010 - 09:44 PM.

[pDNA]

so if i get it right you using your digested, phosphatase-treated vector without ligase and you get a lot of colonies? So your conclusion of undigested vector background seems to be right.

I would suggest doing gel purification of your linearized fragment of the vector ...this should further lower the background.

Concerning your problem that you do not get any colonies when doing the ligation vector+insert:
This could be due to carry over of the phosphatase ...so if you do not inactivate or purify your phosphatase reaction, residual phosphatase can remain and dephosphorylate your insert and prevent it from ligation. This could be one reason why your reaction fails. How do you purified your TSAP reaction?

Regards,
p

[Orangistae]

I did heat-inactivate the TSAP before ligation, so I don't think that would have been an issue. My supervisor suggested that the problem with the ligation could have been over-digestion of the vector, since I did the digest overnight (I haven't tried ligation using the second, 4hr digest), which I hadn't actually heard of before but I guess it might be an explanation. I really want to sort out the digestion problem first though, as it's hard to comment on the ligation efficiency (or lack thereof) when there's so much background from the uncut vector.

Thanks for the suggestion of gel purification, I think I'll have to try that.

[pDNA]

i don't think that it is an issue with overnight digestion.

Also consider that i overdephosphorylation will damage the ends of the vector making it unligatable ...this could be also a problem!

I'm sure you will get that right!

Good luck!

Regards,
p

[HomeBrew]

I agree that you must gel purify both your insert and vector after digestion.  Use a gel to which you have added 0.28 g/L (~1 mM) guanosine (e.g. Sigma G6752, FW 283.24) as a UV protectant to the 1× TAE used to cast the gel and as running buffer during electrophoresis. Stir with gentle heat until dissolved.

I also agree that the dephosphorylation step is frequently a source of trouble -- we use 1/4 to 1/2 the recommended amount, let the reaction proceed for 5 minutes maximum at 37C, and then immediately load the dephosphorylated sample on a gel and turn it on -- we immediately load it on a gel and run it rather than trying to heat-inactivate it, because there is considerable time (relatively) between the sample being at 37C and it reaching the 65C at which the enzyme is inactivated, and during this time all kinds of nasty things can happen to your vector ends.

I would guess that either or both of these two things -- failure to gel purify insert and vector and over-vigorous dephosphorylation, along with ligase buffer problems arising from repeated freeze-thaw cycles and the attendant degradation of the ATP therein -- play a role in a large majority of ligation problems.

[klinmed]

Orangistae, on Apr 24 2010, 06:43 AM, said:

I'm trying to clone some genes into a vector by amplifying them and ligating the PCR products into a SmaI(=blunt-ended cutter)-digested plasmid, but I've been having trouble with it and I've narrowed the problem down to the digestion part. When I run some of the digest on a gel it looks fine, with a single bright band, in contrast to the double band of the undigested vector, but if I transform the digested, unligated vector into E. coli I get heaps of transformant colonies, which seems to indicate that there is a very high background of uncut vector. Can anyone help me work out what the problem is and what I can do about it?

I've tried the digest two times so far, using the same SmaI (Promega, and nowhere near its expiry date), buffer, and DNA sample [which was recently purified and is in EB buffer (i.e. 10mM Tris, pH 8.5) with 1mM EDTA]. The first time, I used 6ul DNA with 2ul each of enzyme and buffer in a 20ul reaction mix and incubated at 25 degrees (SmaI's optimal temp) overnight, before heat-inactivating, treating with TSAP, and transforming 0.125ul of the mixture into E. coli. The second time, I decreased the amount of DNA in the digest to 2ul, incubated the digest for 4hrs, and used 0.25ul for transformation (didn't bother with the TSAP). I got lots (>50) of colonies both times, although somewhat fewer the second time due to the smaller amount of DNA, and when I do the actual ligation and try the transformation with that, I don't get any more colonies compared to using the equivalent amount of 'digested' vector.

Any help or advice would be much appreciated.

I fully agree with the previous wise comments.
You will always get some background when doing blunt ligations. This will be minimized by gel purification of the fragments (very important to keep uv exposure to minimum and/or use guanosine in gels).

It is also easy to overdigest with SmaI.  NEB indicate that even with as low as 10 fold overdigestion they only get 50% religation.  It may be a good idea to kinase a little of your digested/phosphatased vector and self ligate/transform.  This will show if your vector ends and ligation reaction are ok.

To lower background with SmaI ligations a neat trick is to include 0.1 units of the enzyme in your ligation reactions. This will cleave any vector that is able to self-ligate but spare the recombinant vector.

Hope this helps

[Orangistae]

Thanks for the suggestions, everyone; I'll take them into account when I try it again and hopefully it'll work better. Thanks again!