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Cloning dilemma


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#1 rumi84

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Posted 21 May 2009 - 01:43 PM

Hello everyone,

I am trying to clone several insert into a Lenti viral plasmid called pLenti-MP2. These inserts are not PCR cloned but rather obtained from other plasmids and as you all know, when cloning from one plasmid to another you have to make sure that there are same restriction enzymes in both, and in the same order in MCS i.e. 5' and 3' of the insert.

Since I was only able to match one of the two restriction endonuclease from insert to vector, I went ahead and digested the pLenti-MP2 with that enzymes along with CIP to prevent plasmid to religate to itself. The inserts were double digested with the 3'end being compatible with the vector. The problem is how to make the 5'end of the insert ligate with vector since enzymes don't match.

So I guess, the question is "what are my options?"

Thanks for reading this and any suggestion/s is welcome.

rumi84

#2 phage434

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Posted 21 May 2009 - 01:49 PM

Easiest would be to use PCR to amplify your lentivirus plasmid with the 5' end of the primers containing the restriction sites you want. You need to add a half-dozen 5' bases to allow the restriction enzymes to cut effectively. If you treat the PCR reaction with DpnI to cut the template DNA, you can reduce background dramatically.

#3 swanny

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Posted 21 May 2009 - 06:03 PM

Hello everyone,

I am trying to clone several insert into a Lenti viral plasmid called pLenti-MP2. These inserts are not PCR cloned but rather obtained from other plasmids and as you all know, when cloning from one plasmid to another you have to make sure that there are same restriction enzymes in both, and in the same order in MCS i.e. 5' and 3' of the insert.

Since I was only able to match one of the two restriction endonuclease from insert to vector, I went ahead and digested the pLenti-MP2 with that enzymes along with CIP to prevent plasmid to religate to itself. The inserts were double digested with the 3'end being compatible with the vector. The problem is how to make the 5'end of the insert ligate with vector since enzymes don't match.

So I guess, the question is "what are my options?"

Thanks for reading this and any suggestion/s is welcome.

rumi84

IF you can't go down the PCR path (but I really agree with phage434 that it's the simplest thing to do), remember that you only need to reproduce the sticky ends, not the complete site. See what overhang you have from your 5' end digest, and look for an enzyme in the MCS of the target plasmid that generates the same overhang.

Failing that, you can cut the upstream end and fill with Klenow. Now cut with the downstream enzyme and do a blunt-end /sticky end ligation. Not as elegant, perhaps, but you have already chosen to not do the simpler process (PCR).
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#4 rumi84

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Posted 22 May 2009 - 11:31 AM

Hello everyone,

I am trying to clone several insert into a Lenti viral plasmid called pLenti-MP2. These inserts are not PCR cloned but rather obtained from other plasmids and as you all know, when cloning from one plasmid to another you have to make sure that there are same restriction enzymes in both, and in the same order in MCS i.e. 5' and 3' of the insert.

Since I was only able to match one of the two restriction endonuclease from insert to vector, I went ahead and digested the pLenti-MP2 with that enzymes along with CIP to prevent plasmid to religate to itself. The inserts were double digested with the 3'end being compatible with the vector. The problem is how to make the 5'end of the insert ligate with vector since enzymes don't match.

So I guess, the question is "what are my options?"

Thanks for reading this and any suggestion/s is welcome.

rumi84

IF you can't go down the PCR path (but I really agree with phage434 that it's the simplest thing to do), remember that you only need to reproduce the sticky ends, not the complete site. See what overhang you have from your 5' end digest, and look for an enzyme in the MCS of the target plasmid that generates the same overhang.

Failing that, you can cut the upstream end and fill with Klenow. Now cut with the downstream enzyme and do a blunt-end /sticky end ligation. Not as elegant, perhaps, but you have already chosen to not do the simpler process (PCR).


Thanks phage434 and swanny for your suggestions. Re-examining my case, I would say the option with blunt end/sticky end ligation would probably be the best option. Could you share with me the protocol required for producing blunt end using kleenow fragment and the ligation reaction you mentioned? I am new to this and your help would be useful.

Thanks Guys.

rumi84




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