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problem with subcloning, please help!


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#1 paapu

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Posted 12 October 2009 - 09:09 AM

Hai all,

I have been having this cloning problem for a while now...almost three months!

I am trying to replace a fragment of 2 kb from pBR322 vector, have done the same procedure several times before to get various mutants and has worked out so well before .... this will be the last one and it is this one which is causing trouble. here are the details:
the fragment to be replaced is 2 kb in size, has PacI and NotI as the restriction sites on either ends. This is what I do: I digest the plasmid (pBR322) using pacI and NotI (4 hrs digestion, 1ul of each enzyme, 4ug of DNA in 100ul reaction) this releases a 2kb fragment which i clean it out using gel cleaning technique (Qiagen gel purification kit). I have a 2kb fragment with PacI and Not I sites at its ends as a PCR product, also I have cloned the same in a Topo pCR4.0 plasmid. I digest this fragment and purify it. Later I ligate the digested pBR322 with this fragment. Ligation is done overnight at 16C using t4DNA ligase from NEB. I do the transformation in DH10B cells. I am always betrayed...there are no colonies in the plate the next day...it happens all the time....to make sure that the DH10B cells are okay, I did a control transformation...it worked...so the DH10B cells are fine...there is no problem with ligase as people in the lab using it get results!.....I did see something weird in my ligation ...the other day before transformation, I ran a little bit of my ligated product in gel....it was weird...i saw the digested plasmid, my insert and another fragment which was twice the size of my insert...is it possible that the insert got self ligated? or is there something else going on? I am completely frustrated...please help me!

thanks in advance

#2 Flyingbike

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Posted 12 October 2009 - 10:09 AM

Hai all,

I have been having this cloning problem for a while now...almost three months!

I am trying to replace a fragment of 2 kb from pBR322 vector, have done the same procedure several times before to get various mutants and has worked out so well before .... this will be the last one and it is this one which is causing trouble. here are the details:
the fragment to be replaced is 2 kb in size, has PacI and NotI as the restriction sites on either ends. This is what I do: I digest the plasmid (pBR322) using pacI and NotI (4 hrs digestion, 1ul of each enzyme, 4ug of DNA in 100ul reaction) this releases a 2kb fragment which i clean it out using gel cleaning technique (Qiagen gel purification kit). I have a 2kb fragment with PacI and Not I sites at its ends as a PCR product, also I have cloned the same in a Topo pCR4.0 plasmid. I digest this fragment and purify it. Later I ligate the digested pBR322 with this fragment. Ligation is done overnight at 16C using t4DNA ligase from NEB. I do the transformation in DH10B cells. I am always betrayed...there are no colonies in the plate the next day...it happens all the time....to make sure that the DH10B cells are okay, I did a control transformation...it worked...so the DH10B cells are fine...there is no problem with ligase as people in the lab using it get results!.....I did see something weird in my ligation ...the other day before transformation, I ran a little bit of my ligated product in gel....it was weird...i saw the digested plasmid, my insert and another fragment which was twice the size of my insert...is it possible that the insert got self ligated? or is there something else going on? I am completely frustrated...please help me!

thanks in advance


Are you sure of your primers ? I had the same problem with bad primers. Also, dou you have sufficient space around your resctriction sites on your insert ? Some enzymes need some extra bases to "sit" and cut...

You are talking about TOPO, did you try to get your insert out of it and load it onto a gel ?

#3 paapu

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Posted 12 October 2009 - 03:14 PM

Hai all,

I have been having this cloning problem for a while now...almost three months!

I am trying to replace a fragment of 2 kb from pBR322 vector, have done the same procedure several times before to get various mutants and has worked out so well before .... this will be the last one and it is this one which is causing trouble. here are the details:
the fragment to be replaced is 2 kb in size, has PacI and NotI as the restriction sites on either ends. This is what I do: I digest the plasmid (pBR322) using pacI and NotI (4 hrs digestion, 1ul of each enzyme, 4ug of DNA in 100ul reaction) this releases a 2kb fragment which i clean it out using gel cleaning technique (Qiagen gel purification kit). I have a 2kb fragment with PacI and Not I sites at its ends as a PCR product, also I have cloned the same in a Topo pCR4.0 plasmid. I digest this fragment and purify it. Later I ligate the digested pBR322 with this fragment. Ligation is done overnight at 16C using t4DNA ligase from NEB. I do the transformation in DH10B cells. I am always betrayed...there are no colonies in the plate the next day...it happens all the time....to make sure that the DH10B cells are okay, I did a control transformation...it worked...so the DH10B cells are fine...there is no problem with ligase as people in the lab using it get results!.....I did see something weird in my ligation ...the other day before transformation, I ran a little bit of my ligated product in gel....it was weird...i saw the digested plasmid, my insert and another fragment which was twice the size of my insert...is it possible that the insert got self ligated? or is there something else going on? I am completely frustrated...please help me!

thanks in advance


Are you sure of your primers ? I had the same problem with bad primers. Also, dou you have sufficient space around your resctriction sites on your insert ? Some enzymes need some extra bases to "sit" and cut...

You are talking about TOPO, did you try to get your insert out of it and load it onto a gel ?



Thank you for your msg, I had used the same primers before to make other mutations and has worked well, also I did try to digest the insert from TOPO and the size was fine. I forgot to mention about one more thing....I had gotten couple of white colonies sometimes, they are literally white colored and grow in tetracycline, show the size similar to my plasmid DNA but nothing comes out on sequencing. I am kind of frustrated at this stage.

#4 Warren

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Posted 12 October 2009 - 07:03 PM

Hai all,

I have been having this cloning problem for a while now...almost three months!

I am trying to replace a fragment of 2 kb from pBR322 vector, have done the same procedure several times before to get various mutants and has worked out so well before .... this will be the last one and it is this one which is causing trouble. here are the details:
the fragment to be replaced is 2 kb in size, has PacI and NotI as the restriction sites on either ends. This is what I do: I digest the plasmid (pBR322) using pacI and NotI (4 hrs digestion, 1ul of each enzyme, 4ug of DNA in 100ul reaction) this releases a 2kb fragment which i clean it out using gel cleaning technique (Qiagen gel purification kit). I have a 2kb fragment with PacI and Not I sites at its ends as a PCR product, also I have cloned the same in a Topo pCR4.0 plasmid. I digest this fragment and purify it. Later I ligate the digested pBR322 with this fragment. Ligation is done overnight at 16C using t4DNA ligase from NEB. I do the transformation in DH10B cells. I am always betrayed...there are no colonies in the plate the next day...it happens all the time....to make sure that the DH10B cells are okay, I did a control transformation...it worked...so the DH10B cells are fine...there is no problem with ligase as people in the lab using it get results!.....I did see something weird in my ligation ...the other day before transformation, I ran a little bit of my ligated product in gel....it was weird...i saw the digested plasmid, my insert and another fragment which was twice the size of my insert...is it possible that the insert got self ligated? or is there something else going on? I am completely frustrated...please help me!

thanks in advance


Are you sure of your primers ? I had the same problem with bad primers. Also, dou you have sufficient space around your resctriction sites on your insert ? Some enzymes need some extra bases to "sit" and cut...

You are talking about TOPO, did you try to get your insert out of it and load it onto a gel ?



Thank you for your msg, I had used the same primers before to make other mutations and has worked well, also I did try to digest the insert from TOPO and the size was fine. I forgot to mention about one more thing....I had gotten couple of white colonies sometimes, they are literally white colored and grow in tetracycline, show the size similar to my plasmid DNA but nothing comes out on sequencing. I am kind of frustrated at this stage.


pBR322 does not have either a PacI site or a NotI site. Try running an undigested sample of DNA next to your "digested" sample and they should look the same, unless you are getting some star activity. What your trying to do won't work.

Warren..

#5 paapu

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Posted 13 October 2009 - 06:32 AM

Hai, thanks for your reply, we have a linker sequence inside the pBR322 that has these restriction sites, in fact i have cloned into this site ans have done several mutations before at this same sites (Not I and pac I), I am trying to make my final mutant and that is what is giving me the problem.

#6 Warren

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Posted 13 October 2009 - 03:16 PM

Hai, thanks for your reply, we have a linker sequence inside the pBR322 that has these restriction sites, in fact i have cloned into this site ans have done several mutations before at this same sites (Not I and pac I), I am trying to make my final mutant and that is what is giving me the problem.


You have a "linker" or do you have pBR322 with a 2 kb insert that you are using? I can not see how you can cut a 2 kb chunk out of pBR322 without removing either the origin or replication or the tetracycline resistance gene (or part of it). Since you said you are using tetracycline to screen, I assume you are not cutting the gene out? Anyway, one thing I can say is that if you look at your ligation, and you see alot of a band that is double your PCR product size, but not bigger bands (ie, 3, 4 etc of your insert) it suggests only one of the enzymes is cutting your PCR product. This could explain your results.

Warren..

#7 Warren

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Posted 13 October 2009 - 03:19 PM

Hai, thanks for your reply, we have a linker sequence inside the pBR322 that has these restriction sites, in fact i have cloned into this site ans have done several mutations before at this same sites (Not I and pac I), I am trying to make my final mutant and that is what is giving me the problem.


You have a "linker" or do you have pBR322 with a 2 kb insert that you are using? I can not see how you can cut a 2 kb chunk out of pBR322 without removing either the origin or replication or the tetracycline resistance gene (or part of it). Since you said you are using tetracycline to screen, I assume you are not cutting the gene out? Anyway, one thing I can say is that if you look at your ligation, and you see alot of a band that is double your PCR product size, but not bigger bands (ie, 3, 4 etc of your insert) it suggests only one of the enzymes is cutting your PCR product. This could explain your results.

Warren..


PS, there is an easy way to test this, perform a ligation with your digested PCR product only and run it on a gel....

#8 paapu

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Posted 13 October 2009 - 04:57 PM

Thanks Warren, I shall try that too.
Here are some extra info if what I had said wasnt clear. I have a big insert subcloned into pBR322 using a 100bp linker with different unique ezny sites. I am trying to make different mutants at the same area (the 2kb fragment between Not I and Pac I) I have made some mutants doing the same procedure again and again (digesting out the 2 kb fragment and replacing it with another 2 kb fragment that has the mutations in it...actually I change 2-7 aa between the fragments). Every time it was a cakewalk...so as to say....i am working on the final mutant and that is what is giving me the trouble....you might be right that may be one of my enzyme isnt working well....but in that case i wouldnt be able to cut out the 2 kb fragment from my original pBR322....or may be one enzyme site is nt working in the insert fragment and not the enzyme itself...i guess i can sequence the insert to make sure the enzyme sites are okay...hmm...sounds like really a good idea to me....thanks a lot....will try it out and write about it

#9 paapu

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Posted 05 November 2009 - 12:15 PM

Thanks Warren, I finally got the clone....the only thing that I had changed was the primers for the insert, I guess one of the enzyme sites in the primers had been chewed somehow...may be by repeated freezing and thawing or ..whatever the reason (I dont understand) made the primer loose the enzyme site...anyways I ordered new primers with the same sequence and did PCR of the insert, got the insert and cloned in TA vector, digested the insert, digested the plasmid and ligated both. The transformation yielded the desired clone! Thank you




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