Sub-cloning problems!

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Sub-cloning problems!

Postby monkey7 » Mar 10 2010 6:03 pm

Hi everyone,

So basically, I'm sub-cloning a 1.4kb insert from psg5 plasmid into pBluescript, but it doesn't seem to be working and I dont know what else to try.

First of all, I use PCR to amplify, then run product on a 1% agarose gel. I get the correct band at 1.4kb and isolate the DNA using the glass milk protocol. I then ligate it with digested pBluescript (insert doesnt need to be digested, as it has blunt ends). I run both the insert and plasmid on an agarose gel to determine how much insert:plasmid to use..I usually use a 3:1 ratio. I don't check if the ligation works, but it should do.
I then transform into E.Coli competent cells...adding about 1ul of insert to 100ul of competent cells, and using the heat shock protocol. I then spread cells onto LB agar + ampicillin plates containing X-Gal/IPTG for blue/white colony screening. I have done the above set of procedures 5 times and 4 out of 5 times I havent had ANY colonies. The one time that I did get colonies, I got 1-2 blue colonies and 1-2 white colonies per plate, so not much. I extracted the white colonies and then grew them up in universal tubes containing LB Agar+amp at 37C in a shaking incubator overnight. I have carried out both mini preps and midi preps. I first digest the product with BamH1 and Apa1 (as a double digest, using a common buffer). The first time I did the mini prep, I seemed to get a band at 1.4kb, which is what I needed..but I got an extremely large and bright band, and this may be because I used 10ul of dna for the digestion. However, the second time i did it i used only 2ul and seemed to get a band at approx 2kb. I then did a midi prep using a Qiagen kit (all on the same transformation product) and got a band at 2kb instead of 1.4kb and the digestion doesn't seem to have worked very well. Also, my midi prep yields are very low at 20ish ng/ul, but the purity seems ok.

Any ideas? I have a week to get this working!

Thanks!
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Re: Sub-cloning problems!

Postby mchlbrmn » Mar 11 2010 1:12 am

Hi. I have some questions:
The vector is RE digested? With one blunt cutting restriction enzyme? Is it phosphatased to prevent self ligation? Was it then purified? Or, is this a AT or topo vector?
The insert, a PCR product, was not RE cut. Was it kinased to phosphorylate the end so it can ligate?
How competent are the cells? High efficiency cells with 10^9 or more cfu/ug can sometimes help. How many cells did you put on the plate? 100 ul, diluted with SOC to 1 ml, then spun down, might be a bit much for one plate; a toxic effect can reduce bacterial colony size and viability.
From the RE digest do you mean you get 1.4 or 2. kb band plus a vector band? Do they look equimolar: is the vector stronger than the insert?
You used a common buffer. Apai is usually used at 25C; it has a short 1/2 life at 37. It can also be blocked by overlapping dcm methylation (at CCAGG and CCTGG ). Apa is inhibited by >50mM NaCl. BamHI can show star activity in low salt. It might be best to first cut with Apai at 25C in 50mM NaCl, then add 50mM more NaCl and BamHI. Then, perhaps heat inactivate (I may have a memory that someone in the forum said Bam can bind DNA and change gel migration (-but, maybe not). (I'm not saying I know the RE digest is the problem at all, without more details.).
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Re: Sub-cloning problems!

Postby monkey7 » Mar 11 2010 4:48 am

mchlbrmn wrote:Hi. I have some questions:
The vector is RE digested? With one blunt cutting restriction enzyme? Is it phosphatased to prevent self ligation? Was it then purified? Or, is this a AT or topo vector?
The insert, a PCR product, was not RE cut. Was it kinased to phosphorylate the end so it can ligate?
How competent are the cells? High efficiency cells with 10^9 or more cfu/ug can sometimes help. How many cells did you put on the plate? 100 ul, diluted with SOC to 1 ml, then spun down, might be a bit much for one plate; a toxic effect can reduce bacterial colony size and viability.
From the RE digest do you mean you get 1.4 or 2. kb band plus a vector band? Do they look equimolar: is the vector stronger than the insert?
You used a common buffer. Apai is usually used at 25C; it has a short 1/2 life at 37. It can also be blocked by overlapping dcm methylation (at CCAGG and CCTGG ). Apa is inhibited by >50mM NaCl. BamHI can show star activity in low salt. It might be best to first cut with Apai at 25C in 50mM NaCl, then add 50mM more NaCl and BamHI. Then, perhaps heat inactivate (I may have a memory that someone in the forum said Bam can bind DNA and change gel migration (-but, maybe not). (I'm not saying I know the RE digest is the problem at all, without more details.).


Thanks for your response. Yes, the vector is RE digested with EcoR5 and then phosphatased (although I have just been given the vector and haven't done this part myself). However, the insert was not RE cut, nor was it kinased.
How many cells do you suggest plating out?
Yes, I got a 2kb band plus a vector band, but the vector band doesn't appear to be stronger than the insert..although I dont have it on me and need to double-check.
I will definitely try digesting first with Apa1 and then BamH1, modifying the buffers..I was thinking this myself yesterday, so thank you !

Thanks, once again.
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Re: Sub-cloning problems!

Postby relaxin » Mar 11 2010 9:24 am

You have ligation problem. Your insert needs to be phosphorylated with T4 polynucleotide kinase and ATP, otherwise it will not be ligated to the dephosphorylated vector. Also, if you are not using proof-reading polymerase (such as Pfu or Pfx), you need to polish the ends of the insert with T4 DNA polymerase and dNTPs.
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Re: Sub-cloning problems!

Postby r.rosati » Mar 11 2010 9:48 am

Wait wait, hold the horses! Heheh :)

I think you're facing several problems here, and misinterpreting your results.

1) Results: did you check the digested plasmid along with an undigested aliquot? I ask because, if you didn't, then a possibility is that the RE didn't cut at all, your 2kb band being supercoiled plasmid DNA. In an empty vector. And brighter band = faster band, i.e. if you overload the gel, then the band will run faster and look "shorter".

2) problems:
- you're trying to have a ligase join an unphosphorylated PCR product to a dephosphorylated vector. Unless you used 5'-phosphate modified primers, think about it and you'll realize: it won't work. If it does work, it's either because one vector or insert molecule got nibbled by DNAses, exposing a 5'-phosphate, or because one lucky vector survived the phosphatase treatment with its 5'-phosphates intact. In the first case, you don't get what you wanted. In the second case (the "Highlander" vector) you get the right plasmid, but the chances of the vector surviving the phosphatase and not religating to itself are very, very dim.
- as a side note, you're using someone else's vector. As they said in Paranoia RPG, Stay alert, trust no one, and keep your laser handy! A happy lab is one where people trust each other, but don't need to. If you kinase the PCR product and things still don't work, then think - is this batch of "ligation-ready" vector a recent one? has it been used recently with good results? Can you take another aliquot of the prepped vector and digest & dephosphorylate it yourself? Of course, if you only have one week, it''d be best to just prepare your batch and test both.
-as another side note, are you using a proofreading polymerase (= blunt ends?)
-as a last side note, are your competent cells ok? Tested for efficiency?


(aw, relaxin beat me - I should work less and post more, heheh :wink: )
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Re: Sub-cloning problems!

Postby monkey7 » Mar 11 2010 10:00 am

Thank you relaxin and weidomen!

It looks like I definitely have ligation issues. Somebody else in the lab simply gave me the vector (pBluescript) and said that she digested it with EcoRV to give blunt ends and then phosphatased it. At no point do I phosphorylate my insert sample! I have just been isolating my DNA post-PCR (for which I used Phusion polymerase) and then ligating straight away. Oops. So do I treat my insert with T4 kinase and ATP and then proceed with ligation? Should I check that the ligation has worked prior to transformation?
We are getting higher efficiency competent cells, so perhaps that will make a difference.

Thanks :D
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Re: Sub-cloning problems!

Postby r.rosati » Mar 11 2010 10:12 am

Yes, just remember to inactivate the T4 kinase by heat before the ligation.
I don't check ligations (you need to load quite a bit of DNA to see it on gel). But, of course, it might be interesting in your case. I guess it's up to you.
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Re: Sub-cloning problems!

Postby relaxin » Mar 12 2010 12:42 pm

monkey7 wrote:Thank you relaxin and weidomen!

It looks like I definitely have ligation issues. Somebody else in the lab simply gave me the vector (pBluescript) and said that she digested it with EcoRV to give blunt ends and then phosphatased it. At no point do I phosphorylate my insert sample! I have just been isolating my DNA post-PCR (for which I used Phusion polymerase) and then ligating straight away. Oops. So do I treat my insert with T4 kinase and ATP and then proceed with ligation? Should I check that the ligation has worked prior to transformation?
We are getting higher efficiency competent cells, so perhaps that will make a difference.

Thanks :D


I am intrigued by your username. Years back, a fellow postdoc from Chile (a protein chemist) learned some molecular biology techniques from me and said, "Oh, it was so easy, I can train monkeys to do it!". I just wonder if he has finally suceeded.

Not to make fun of your username. Just want to cheer you up when your experiment does not work. :D
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Re: Sub-cloning problems!

Postby monkey7 » Mar 14 2010 8:01 pm

relaxin wrote:
monkey7 wrote:Thank you relaxin and weidomen!

It looks like I definitely have ligation issues. Somebody else in the lab simply gave me the vector (pBluescript) and said that she digested it with EcoRV to give blunt ends and then phosphatased it. At no point do I phosphorylate my insert sample! I have just been isolating my DNA post-PCR (for which I used Phusion polymerase) and then ligating straight away. Oops. So do I treat my insert with T4 kinase and ATP and then proceed with ligation? Should I check that the ligation has worked prior to transformation?
We are getting higher efficiency competent cells, so perhaps that will make a difference.

Thanks :D


I am intrigued by your username. Years back, a fellow postdoc from Chile (a protein chemist) learned some molecular biology techniques from me and said, "Oh, it was so easy, I can train monkeys to do it!". I just wonder if he has finally suceeded.

Not to make fun of your username. Just want to cheer you up when your experiment does not work. :D


Ha, thank you :)
Well, my supervisor told me that I shouldn't be dephosphorylating my vector in the first place (which is pBluescript). He told me to just repeat the entire process again and then digest the miniprep product with a RE that should cut into the insert, but not the vector.
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Re: Sub-cloning problems!

Postby relaxin » Mar 14 2010 9:42 pm

Your supervisor is absolutely wrong. BlueScript vector cut with EcoRV produced a blunt-end. Without dephosphorylation, the insert cannot complete with the self-ligation of the vector. It is easier to treat the PCR fragment with T4 polynucleotide kinase than to screen many white colonies, if you are lucky to have a few (most of the time, even white colonies have no insert).
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Re: Sub-cloning problems!

Postby monkey7 » Mar 27 2010 7:10 am

Hello,
It's me again. I'm trying to analyse my agarose gel of mini prep products (following ligation and transformation) with a bit of difficulty. I used 2 RE which would cut into the insert and vector and then 2 RE which would only cut into the insert. I got the same result. I'm expecting a band at 1.4 kb, but instead I get : digested = 2-3 kb (just the vector?), undigested = 1.5kb, large band at 2kb, bands at 10kb and above (supercoiled dna??). Any ideas? Also, is it possible that the insert is no longer 'in frame'? My supervisor used this term and said perhaps it's no longer in frame, and that's why it's not being digested by the REs, but I'm not too sure what exactly he means by that...

Thanks :D
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Re: Sub-cloning problems!

Postby relaxin » Mar 27 2010 10:09 am

I do not quite understand your cloning strategy. As I understood, you amplify the insert and cut with two RE (presumably at the sits encoded by the primers). Then you ligated the insert to the vector digested with the same RE (without dephosphorylation, as suggested by the supervisor). Then the insert should be "in frame" with the vector and should be re-cut by the same RE. However, there are chances that the vector self-ligated via blunt-end ligation, because the sticky ends are removed by nonspecific nuclease activity. Then you cannot recut the insert from the miniprep.

I would suggest to screen the colonies by PCR using primers based on the vector sequence flanking the cloning sites.
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Re: Sub-cloning problems!

Postby monkey7 » Mar 27 2010 12:02 pm

I did not cut the insert with anything, but simply digested the vector with EcoRV in order to create blunt ends. I'm not sure why he favoured that over a cohesive end ligation.
I won't be carrying on with this work, as my time in the lab has come to an end. I'm just trying to make sense of the restriction analysis for my report..
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Re: Sub-cloning problems!

Postby relaxin » Mar 28 2010 10:31 am

monkey7 wrote:I'm expecting a band at 1.4 kb, but instead I get : digested = 2-3 kb (just the vector?), undigested = 1.5kb, large band at 2kb, bands at 10kb and above (supercoiled dna??). Any ideas? Also, is it possible that the insert is no longer 'in frame'? My supervisor used this term and said perhaps it's no longer in frame, and that's why it's not being digested by the REs, but I'm not too sure what exactly he means by that...


It looks like your digested band is just the vector, there is no insert (1.4 kb). I do not know what the 1.5 kb band in undigested DNA. The 2 kb and is the undigested vector. The 10 kb band is the bacterial genomic DNA. Since the insert was ligated to vector via blunt-end, and the RE is within the insert, so there is no problem with the frame. I do not know what your supervisor meant either.
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