Hi Belete,

Thank you for contacting me. The first thing I would want to know is whether or not the restriction enzymes that you used have effectively cut the PCR product. Usually additional sequence is required flanking the restriction site so that the enzymes can cut effectively. I have checked on the ones you outline above and 3bp should be fine for both of those enzymes. When you ligated the 59bp, 61bp and 713bp sequences were these also cut with the same enzymes?

The other question that I have is what insert:vector ratios are you using in your ligation? You have to calculate the insert and vector concentrations in terms of nmoles of ligation ends in the reaction (using 660 as the molecular weight of a single bp) and then set up 3 ligations at 3:1, 1:1 and 1:3 insert:vector ratios.

Have you tried this?

Many thanks and I look forward to hearing back from you Belete.

Many thanks,

Robert

Robert Deyes

Technical Services Scientist

Promega Corporation

Tel 1 800 356 9526 Ext 1366 (USA)

Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)

http://www.promega.com

EMail: [email protected]

Hi Belete,

Thank you for contacting me. The first thing I would want to know is whether or not the restriction enzymes that you used have effectively cut the PCR product. Usually additional sequence is required flanking the restriction site so that the enzymes can cut effectively. I have checked on the ones you outline above and 3bp should be fine for both of those enzymes. When you ligated the 59bp, 61bp and 713bp sequences were these also cut with the same enzymes?

The other question that I have is what insert:vector ratios are you using in your ligation? You have to calculate the insert and vector concentrations in terms of nmoles of ligation ends in the reaction (using 660 as the molecular weight of a single bp) and then set up 3 ligations at 3:1, 1:1 and 1:3 insert:vector ratios.

Have you tried this?

Many thanks and I look forward to hearing back from you Belete.

Many thanks,

Robert

Robert Deyes

Technical Services Scientist

Promega Corporation

Tel 1 800 356 9526 Ext 1366 (USA)

Tel 1 608 274 4330 Ext 1366 (Long Distance, Toll Paying)

http://www.promega.com

EMail: [email protected]

Hi Belete,

A quick way to give some idea of toxicity to the cloning host (assuming you are doing a bacterial transformation) would be to blunt clone your insert into Puc19 cut with sma1 first. I have done this with difficult clones, and found that I got recovery, except when analysing the recombinants, they all had the insert in the reverse orientation, suggesting they were toxic when transcribed. It also helps if you are PCR amplifying your insert to add the RE sites, as you have a ready supply of your insert, and know that your digests are working as you can see the fragment released from the Puc vector. The relatively large size of your insert compared to Puc19 means you could easily separate them on a gel.

Does the nature of the protein encoded by the insert suggest it may be toxic ?

You seem to have quite a large vector to clone into, is it anything special that may be causing problems.

Hope this helps

Most often these cases deal with ligation/cloning inefficency. 5.3 KB is a relatively large insert, and a 10KB vector is also on the large size for standard cloning. What Robert has suggested is a good start to resolving your issues. Other things that you can try are to use a host cell that is designed to handle and stabilize larger constructs. You can also increase or change your ligation times and/or incubation temperatures. Definitely make sure you try the variable ligations based on molar ratio's, that usually does the trick for me.

Thank you all for your suggestions. Just to be more specific, insert was PCR amplified with primers with AvrII and XmaI/SmaI sites. It was gel purified and its two ends were sequenced to make sure that the restriction sites are there. Enough bps were added at the ends of each restriction sites, sequentially digested overnight with XmaI and for four hours with AvrII (as AvrII has a star activity) . Vector was also digested with the same enzymes as that of insert and treated with CIP.

I'm making a construct, and so far cloned three fragments of different restriction sites to the vector very easily. This one seems tricky.

Ligating the blunt ends of the insert to different vector and cutting it out using the two restriction sites is a best way to go to get a higher yield, as David suggested.

Still playing with different ratios of inset:vector, ligation temp and more suggestions are welcome.

Thanks Thomas for the suggestion, I've managed to get it done in a different way. There is a unique restriction site (NheI) halfway in the insert and I designed a reverse primer of : bps homologous to insert + NheI + 3ps + CCC. In the first round of PCR, Insert was amplified by this reverse primer and the original forward primer whose end was AvrII. The amplified fragment was digested by AvrII; vector by AvrII and SmaI. Sticky/blunt ends ligation was carried out at room temperature. This introduces NheI site in the vector. In the second round of PCR, I have used primer whose ends were NheI/SmaI to amplify the second half of the insert. In both cases I have got less than 6 clones after transformation and two of them were positive. Fair play!