Is my insert present??? - (Oct/28/2009 )

Hi all...new the forum.

I have been trying to clone MSH3 into pCS2+MT and last week, I think we were able to get it. I am using the restriction sites EcoRI and XhoI.

When i single digest the possible MSH3/pCS2+MT construct, the size is close to the 8kb band (1kb Tri Dye) which is close (MSH3 = 3.4kb pCS2+MT = 4.35kb).

However, when i double digest the plasmid, i get one band that is the size of the empty vector but i get a 2nd band that is about 2kb, much smaller than the needed 3.4kb.

I've repeated this twice with the same outcomes; appropriate single-digested size but smaller double-digested insert size. (or at least what i think is the insert) There are no other bands present on the gel.

What is going on? Any help is appreciated. Thanks.

Have you tried single digestions with each of the enzymes? On double digest, could the smaller band be a doublet?

HomeBrew on Oct 29 2009, 07:13 AM said:

how can a doublet give a much smaller band!!! me confused...
and ya you can try the single digests with both the enzymes separately...

Pradeep Iyer on Oct 28 2009, 10:38 PM said:

I already did single enzyme digests. They both gave me the ~8kb band which is what the insert + vector is approximately. I explained that in the original post.

I already made sure that the insert does not contain any internal restriction sites to those i used to clone into the vector.

I even ran a gel to see the size of my insert after double digestion before ligation and it is the correct size...it falls between 2 and 3 kb.

rocketfan86 on Oct 29 2009, 10:24 AM said:

may be you were not clear so we thought you did only one..
may be you are incubating it for a long enough time for some star activity and so the band size.
Try optimizing the restriction reaction!!! or may be do a single difest with the first enzyme and follow it up with the second instead of doing it both simultaneously!!!
Best luck!

rocketfan86 on Oct 29 2009, 09:24 AM said:

hey, i m sorry , im confused....but I think u initially said that ur insert, ie, MSH3 is 3.4 kb. Now, u mention dat it is b/w 2 and 3 kb....how is dat possible????

Pradeep Iyer on Oct 28 2009, 11:38 PM said:

It was not clear to me originally that rocketfan86 had done single digests with both enzymes. My thinking was that perhaps rocketfan86's insert was being cleaved in half by one of the enzymes, thus turning a 3.4 kb insert into two 1.7 kb fragments, which might appear as a single band (a "doublet") running at approximately 2 kb.

I suppose this still might be possible, depending on how rocketfan86 "made sure that the insert does not contain any internal restriction sites to those i used to clone into the vector": if it was done by sequencing the clone, it's not possible (barring a sequencing error), but if it was done by analyzing a publicly deposited sequence of the insert, there could be a site in the cloned insert that's not in the deposited sequence.

The single digest with the offending enzyme would produce two fragments on digestion instead of one, and the vector band would be decreased in size by the amount of the smaller second band.

Since it is impossible to lose mass, and there is apparently some DNA missing in the double digest, there's either a doublet hiding somewhere, or the sizing estimates in the double digest are wrong. The only ways I see to figure this out is to sequence the insert and see what you've got, or to run the double digest under different gel concentration conditions and with a different ladder to see if there's a sizing estimate error...

Why not try and do a PCR

try out a colony PCR in a 20ul solution with primers flanking the 3' and the 5' end of your gene
if the product is the correct size then ur insert is fully there!

Deezoo on Oct 29 2009, 08:30 AM said:

Another good idea! I don't know why I didn't think of that (we do it all the time) -- not enough coffee this morning, I guess...

HomeBrew on Oct 29 2009, 08:12 AM said:

I did a pcr last night using the primers i amplified off of the original vector the MSH3 gene came in. I used my construct to amplify off of as well as the original MSH3/pCR4-TOPO it came in. I'll be running the gel this morning.

It is possible that one of the enzyme may cleave an internal site but i didn't see any additional sites for EcoRI and XhoI. It could be star activity. I double digested originally at 3 hours and this last time 1 hour and still got the same band.

I'll let you know what information comes from the gel.

Thanks for all your suggestions!