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strange cloning problem

ligation cloning

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#1 comfy_numb

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Posted 29 December 2011 - 08:34 AM

Hi Guys,

I have a strange cloning problem. I am trying to clone a 2800 bp insert into 4203 vector.

I have verified each step of the cloning process with close attention. After PCR product, I purified and digested vector and insert with the same restriction digests, CIP treated the vector for an hour at 37C. I verified the correct band size with agarose gel. After digestion, ran in the gel, gel extracted and ran them again to quantification before ligation.

Did ligation at 16c overnight. Transformed with DH5Alpha cells. The negative control , linearized vector gives no colonies. The 1:2 and 1:3 gives lot of colonies. but whether doing colony PCR or minipreps, I don't seem to have insert in any of my colonies. I have gone back to verify each step of the clonign process. After screening close to 40 colonies, I am surprised I don't have a single positive clone. It really seems strange that this has not worked in the last step.

If you guys have any insight. Please share.

Frustratingly yours,

Comfy

#2 phage434

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Posted 29 December 2011 - 06:23 PM

I would try reducing the complexity of your protocol. Skip the CIP treatment. Skip the gel extraction. You should be able to do this ligation without either of these steps, and they are likely causes of problems. Are you cutting with one or two enzymes? Have you added 5' extensions on your primers to allow the PCR product to be cut? Cutting with two enzymes, with different enzymes on opposite ends of the PCR product is a strategy that usually works. Less handling and simpler protocols have less opportunity for problems. I would do pcr, clean up (necessary), then mix the clean pcr product with the vector, digest them together with the two enzymes, heat kill the enzymes (choose some that can be heat killed), mix with ligation buffer, ligase, and transform.

#3 lamaksha77

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Posted 30 December 2011 - 09:18 AM

I would try reducing the complexity of your protocol. Skip the CIP treatment. Skip the gel extraction. You should be able to do this ligation without either of these steps, and they are likely causes of problems.


If he is using a single RE, then skipping CIP would give even more empty vector colonies, and going straight to the ligation without purifying the restriction digest could interfere with the ligation reaction (ligases are often pretty salt sensitive). Phage434 is fully right in saying that cutting down on the steps would reduce the risk of errors, but personally, I would stick to OPs protocol.

I think you might want to increase the vector:insert ratio though. We routinely use 1:8 in our lab. Secondly, the fact that you are getting empty vector colonies even after CIP treatment means that either your CIP treatment isn't working properly, or your RE is not fully cutting the vector - this then results in uncut vectors, which are obviously not affected by the CIP treatment. which transform DH5a to give the false positives One way of checking this is to do a RE digest of the vector, and then run it on a gel. If you see two bands, your RE is not fully cutting the vector, and you might have to incubate for longer/use a more appropriate buffer....

#4 pDNA

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Posted 01 January 2012 - 02:52 AM

i would do quality control of restriction digest steps ...both for vector and insert ...this means setting up a ligation reaction with solely insert or vector (approx. 100-200 ng ...so that you can see something on the gel) and run it on an agarose gel ...you should get a kind of ladder (dimer, trimer, ...multimers) ...indicating that vector and insert are properly cut on both sides ...if you just get dimers you will know that only one of your enzymes did cut. I always do this in the course of trouble shooting.

What enzymes are you using?

If you don't get positive clones repeatedly this could be an indication that your insert is somehow toxic (is there an bacterial promoter upstream?).

Good luck!

Regards,
p

#5 comfy_numb

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Posted 02 January 2012 - 01:34 PM

Hey guys,

Thank you for your responses. I think both my enzymes do cut and I have already verified that running on a gel with the vector.

I am using two REs. Xho I and Sal I. I know some of you may say SAL I has some problems associated with cutting. With PCR, i am putting these two restriction enzyme sites in my DNA. I don't think vector has a problem because after gel extraction, I use both xho I and SAL I separately with other two enzymes to see i get correct fragments, so i know these contain the right sizes and both the enzymes are cutting at least in the vector.

One thing I cant verify if the insert has those restriction enzyme sites after pcr reactions. I still don't understand CIP not working because the vector that is CIP treated after digestion doesn't give any colonies on plate. but the ones with vector/insert after ligation do give the colonies. Do you think the problem could be in this PCR reaction when trying to put in restriction enzyme sites in the insert?

#6 phage434

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Posted 02 January 2012 - 02:09 PM

The problems with SalI have largely to do with cutting at the ends of PCR products, not in cutting vector DNA. Make absolutely sure you have many (8-10) bases of 5' DNA past the SalI site on your PCR primer, so that the PCR product being cut with SalI looks as much as possible like a normal piece of DNA. If necessary, you can verify that the PCR product is being cut by doing a test digestion and ligation. PCR cleanup your band, then digest with SalI only (or with XhoI only). After heat kill or cleanup, ligate with normal T4 DNA ligase buffer (not quick ligase). Heat kill the ligase, then run the product on a gel. You should see two bands -- a single and double length band. The ratio of these two tells you how efficient your cutting and ligation at that site is. If you see no double length fragment, you have found your problem.

#7 comfy_numb

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Posted 10 January 2012 - 07:37 AM

Thanx veteran.

however, the vector DNA is also a pCr product as I have put Xho I and Sal I sites on it as well, and verified by cutting in parallel XhO i and SAL i with another enzyme to get desired fragment. One could still argue that SAL i probably didn't cut at the pcr product. I will follow your advice and see if it works.

Thanx

#8 comfy_numb

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Posted 11 January 2012 - 01:53 PM

Hi Phase 434,

I actually did a test digestion and ligation with Sal I and I get the double length fragment like you mentioned.

I am redoing everything from fresh hoping I was just plain unlucky the first time around. I'll keep you posted.

#9 Jash

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Posted 13 March 2012 - 01:18 AM

Hi Comfy... I am also frustrating with the cloning stuff... well i am trying to clone 2294, 1973 and 1860 bp insert into approx 8500 vector...
for 2294 insert, i use Xma1 (isoschizomer of Sma1) in both forward and reverse primer. I am unable to clone.. i should dephosphorylate the vector.. could you please suggest anything on it..

For the 1973 bp inser, i cud not get the pcr using the high GC buffer as well as Phusion.. suggest me something...
For 1860 i go the PCR, tried to clone in the 8500 vector and trasnformed in DH5alpha.. (used 15 micro litre in 200 micro liter DH5alpha cells) and then used 200 on the ampicillin plates. sprayed IPTG+X-gal (48 micro litre). i got many colonies.. but i cud not differentiate between the blue and white becasue i did not have any blue colony.. can u suggest something..
then i proceeded with the colony PCR i got the correct results and sent good clones for sequencing... but at the first go i was said to reculture the samples..
also when i digested the 1973 and 1860 insert usning Not1 and BglII i did not get any result.. althogh i cud get the vector band but no fragment band.. or any othe band... will you please suggest me something...

COmments awaited from various researchers and scientists.. or biologists...
thank u
Comfy.. and keep going..

Hi Guys,

I have a strange cloning problem. I am trying to clone a 2800 bp insert into 4203 vector.

I have verified each step of the cloning process with close attention. After PCR product, I purified and digested vector and insert with the same restriction digests, CIP treated the vector for an hour at 37C. I verified the correct band size with agarose gel. After digestion, ran in the gel, gel extracted and ran them again to quantification before ligation.

Did ligation at 16c overnight. Transformed with DH5Alpha cells. The negative control , linearized vector gives no colonies. The 1:2 and 1:3 gives lot of colonies. but whether doing colony PCR or minipreps, I don't seem to have insert in any of my colonies. I have gone back to verify each step of the clonign process. After screening close to 40 colonies, I am surprised I don't have a single positive clone. It really seems strange that this has not worked in the last step.

If you guys have any insight. Please share.

Frustratingly yours,

Comfy



#10 Jash

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Posted 13 March 2012 - 01:19 AM

let me make clear.. for 2294 insert RE is Xma
For 1973 and 1864 i am using Not1 in the forward and BglII in the reverse..


Hi Comfy... I am also frustrating with the cloning stuff... well i am trying to clone 2294, 1973 and 1860 bp insert into approx 8500 vector...
for 2294 insert, i use Xma1 (isoschizomer of Sma1) in both forward and reverse primer. I am unable to clone.. i should dephosphorylate the vector.. could you please suggest anything on it..

For the 1973 bp inser, i cud not get the pcr using the high GC buffer as well as Phusion.. suggest me something...
For 1860 i go the PCR, tried to clone in the 8500 vector and trasnformed in DH5alpha.. (used 15 micro litre in 200 micro liter DH5alpha cells) and then used 200 on the ampicillin plates. sprayed IPTG+X-gal (48 micro litre). i got many colonies.. but i cud not differentiate between the blue and white becasue i did not have any blue colony.. can u suggest something..
then i proceeded with the colony PCR i got the correct results and sent good clones for sequencing... but at the first go i was said to reculture the samples..
also when i digested the 1973 and 1860 insert usning Not1 and BglII i did not get any result.. althogh i cud get the vector band but no fragment band.. or any othe band... will you please suggest me something...

COmments awaited from various researchers and scientists.. or biologists...
thank u
Comfy.. and keep going..


Hi Guys,

I have a strange cloning problem. I am trying to clone a 2800 bp insert into 4203 vector.

I have verified each step of the cloning process with close attention. After PCR product, I purified and digested vector and insert with the same restriction digests, CIP treated the vector for an hour at 37C. I verified the correct band size with agarose gel. After digestion, ran in the gel, gel extracted and ran them again to quantification before ligation.

Did ligation at 16c overnight. Transformed with DH5Alpha cells. The negative control , linearized vector gives no colonies. The 1:2 and 1:3 gives lot of colonies. but whether doing colony PCR or minipreps, I don't seem to have insert in any of my colonies. I have gone back to verify each step of the clonign process. After screening close to 40 colonies, I am surprised I don't have a single positive clone. It really seems strange that this has not worked in the last step.

If you guys have any insight. Please share.

Frustratingly yours,

Comfy







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