8 replies to this topic

[HawkPidgeon]

Hello, everyone. I'm doing a study in genetics at my school, and I've run into a bit of a roadblock.

Part of my study involves performing subcloning. I understand that a gene of interest must be isolated from a parent vector before it can be ligated into a new vector, and that the ends of the gene of interest and the vector you're trying to insert it into have to have been cut by the same restriction enzymes.

For example, if you have plasmid A and plasmid B, and you want to take a gene from plasmid A and insert it into plasmid B, you have to do a restriction digest to cut the gene away from plasmid A, as well as a restriction digest -- using the same plasmids as you used with plasmid A -- to open plasmid B such that the gene can then be inserted.

So I'm wondering: What do you do when subcloning if your gene of interest and your vector don't have any complementary restriction sites?

Thank you.

[phage434]

Before PCR, you had a problem.  With PCR reactions, you can add restriction sites to either the insert or the vector  backbone as necessary to make your cloning work easy.  This has eliminated much of the utility of multiple cloning sites, although most users don't think about using PCR to add restriction sites to vector backbones, for some unknown reason.

[HawkPidgeon]

phage434, on 16 April 2012 - 01:19 PM, said:

Before PCR, you had a problem.  With PCR reactions, you can add restriction sites to either the insert or the vector  backbone as necessary to make your cloning work easy.  This has eliminated much of the utility of multiple cloning sites, although most users don't think about using PCR to add restriction sites to vector backbones, for some unknown reason.

That makes a whole lot of sense, phage434. I'm reading about that process now. Thank you very much.

Edited by HawkPidgeon, 16 April 2012 - 01:44 PM.

[bob1]

What people did was use adapters which contained the appropriate restriction sites, these were ligated onto the end of the sequence of interest after it had been removed from the parent plasmid, and then cut, and the sequence then ligated into the plasmid.

[HawkPidgeon]

bob1, on 16 April 2012 - 05:37 PM, said:

What people did was use adapters which contained the appropriate restriction sites, these were ligated onto the end of the sequence of interest after it had been removed from the parent plasmid, and then cut, and the sequence then ligated into the plasmid.

You're talking about before PCR came around? How did they remove the sequence of interest from the plasmid in the first place without PCR or a restriction site? (I'm just asking out of curiosity now.)

[bob1]

HawkPidgeon, on 16 April 2012 - 05:44 PM, said:

You're talking about before PCR came around? How did they remove the sequence of interest from the plasmid in the first place without PCR or a restriction site? (I'm just asking out of curiosity now.)

The question asks about the absence of complementary restriction sites - you still need restriction sites to cut out the sequence of interest.

[Do1ngP3e]

Back in the olden days (1990s) we had this great little trick called Blunt end cloning where by we used Mung Bean nuclease or Klenow to polish up the ends so that there was no overhang on the vector..... or the insert.  After removing the Phosphate groups from the vector so it couldn't self ligate, you could ligate in anything in you wanted -- though putting seqeuences in in frame was a little trickier....

[HawkPidgeon]

Do1ngP3e, on 23 April 2012 - 10:47 AM, said:

Back in the olden days (1990s) we had this great little trick called Blunt end cloning where by we used Mung Bean nuclease or Klenow to polish up the ends so that there was no overhang on the vector..... or the insert.  After removing the Phosphate groups from the vector so it couldn't self ligate, you could ligate in anything in you wanted -- though putting seqeuences in in frame was a little trickier....

After talking to a few people about it, this is what I will be doing. Thank you!

[memari]

HawkPidgeon, on 16 April 2012 - 12:58 PM, said:

What do you do when subcloning if your gene of interest and your vector don't have any complementary restriction sites?

Thank you.

Take a look in NEB site and check for COMPATIBLE ENDS for RE Sites. For example these are Restriction enzymes which make compatible end with BamHI
http://www.neb.com/n...radSearchType=0
or you can do a PCR or bring out the sequence of yours interest. then you can insert it to a TA cloning Vector.
https://en.wikipedia...wiki/TA_cloning
http://www.protocol-...ning/index.html
and after transfecting it to E coli and multipying it, you can purify it and cut it again. This vector has its own Multiple Cloning sites.
and then inserting your seuence to another vector.

Babak