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Is my insert present???


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#1 rocketfan86

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Posted 28 October 2009 - 12:46 PM

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.

#2 HomeBrew

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Posted 28 October 2009 - 05:43 PM

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

#3 Prep!

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Posted 28 October 2009 - 07:38 PM

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


how can a doublet give a much smaller band!!! me confused...
and ya you can try the single digests with both the enzymes separately...
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#4 rocketfan86

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Posted 28 October 2009 - 07:54 PM

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


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


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.

#5 Prep!

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Posted 28 October 2009 - 09:42 PM

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.


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!
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#6 Vini

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Posted 28 October 2009 - 10:09 PM

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


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


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.



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???? ;)

#7 HomeBrew

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Posted 29 October 2009 - 04:22 AM

how can a doublet give a much smaller band!!! me confused...


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...

#8 Deezoo

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Posted 29 October 2009 - 04:30 AM

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!

#9 HomeBrew

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Posted 29 October 2009 - 05:12 AM

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!


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...

#10 rocketfan86

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Posted 29 October 2009 - 05:52 AM

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!


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...


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!

#11 HomeBrew

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Posted 29 October 2009 - 12:37 PM

It's an interesting problem, rocketfan86 -- let us know how it progresses towards a solution...

BTW, welcome to the BioForums!

#12 rocketfan86

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Posted 29 October 2009 - 01:00 PM

It's an interesting problem, rocketfan86 -- let us know how it progresses towards a solution...

BTW, welcome to the BioForums!


Thank you HomeBrew!

So i ran PCR on 1% Agarose Gel and the positive control (MSH3/pCR4-TOPO) worked but the construct did not. I need to figure out how to make it work.

Right now, i will run another gel to look at another digestion of MSH3/pCS2MT. I have used another restriction site (BamHI) and an original XhoI to see if the insert is present so the size of the insert should be ~3.7kb. If that doesn't work, i guess i can sequentially digest EcoRI and XhoI to see if that makes a difference.

Keep you posted.

#13 HomeBrew

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Posted 29 October 2009 - 01:59 PM

Let's back up a bit... What was the nature of your transformation? Did you get a lot of colonies, or only a few?

At some point, it makes more sense to just start over and re-clone it.

#14 rocketfan86

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Posted 29 October 2009 - 06:49 PM

Let's back up a bit... What was the nature of your transformation? Did you get a lot of colonies, or only a few?

At some point, it makes more sense to just start over and re-clone it.


Nature? Just thawing DH5-alpha E. coli on ice , put the ligated plasmid/insert into the bacteria, flick tube, rest on ice for 15 minutes, heat shock for 30 seconds at 42C, put back on ice for 2 then plate. It was getting late so i skipped the adding of SOC media and incubating in 37C shaker to recover.

I got about 60 colonies. + ligation control got about 23 colonies but - ligation control got 20 colonies and -/- ligation/transformation got 12 colonies.

Makes me think there is contamination or incomplete digestion.

Probably need to make some new Amp+LB plates.

For this construct it took me 9 times to finally get one colony that looked like it contained the insert...i guess i'm losing hope fast.

Trying the other double digestion with another enzyme and XhoI showed a band the size of an empty vector. Needless to say I was devistated. To top that off, the other construct i've been trying to make, EXO1 and pFLAG-CMV2, once again showed only empty vector. Very frustrating indeed and still wondering i i decided to be a graduate student.

#15 almost a doctor

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Posted 30 October 2009 - 02:03 AM

Hi rocketfan86, dont despair just yet. Science (and cloning in this case) can be a tricky matter but is rewarding in the end.

From reading all the posts I understand you are trying to prove a particular mini-prep contains your insert. In my opinion I think you are getting stuck in the wrong stage, I totally agree with homebrew that sometimes starting all over is better, it might save you more time than you initially imagine.

In one of the latests posts you said you have 22 colonies in you ligation, but 20 in the negative control... it looks to me like the problem is your ligation then and you are trying to prove your positive is positive when in fact is empty vector (quite frankly I think you have enough proof to say is a fail, and is just empty vector).

Could you post your full protocol: how do you obtain the insert? how do you prepare your vector? what's your ligation protocol? what's your transformation protocol? I think we would be able to help you much better if you tell us all this information and we can walk thru the problem step by step.
Dont take this the wrong way, but if your other construct (EXO1 and pFLAG-CMV2) isn't working better, you might be missing some point and we might be able to help define it.

Hope this helps.




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