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Blunt end cloning

help please!

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12 replies to this topic

#1 Clare

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Posted 19 June 2014 - 01:36 AM

Hi everyone,

 

I hope someone can help me out! I normally have no trouble with cloning but for some reason my latest efforts just ain't working :/

 

I need to replace the promoter of one vector with another. RE sites to use are very few so this is what I have been trying to do:

 

1. Remove existing promoter with Xbal and NotI (get expected sizes so all good here).

2. Remove new promoter with StuI and MluI (again, expected sizes).

3. Gel purify bands.

4. Blunt ends with Mung Bean Nuclease and purify.

5. CIP-treat vector and purify.

6. T4 Kinase treat insert.

7. Ligate 16degC ON.

 

I have also tried a few variations of not CIP-treating/T4 Kinase treating but absolutely nothing has worked :/

 

Any advice would be greatly appreciated!

 

Clare



#2 bob1

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Posted 19 June 2014 - 02:40 AM

The easiest way to do this is to amplify the new promoter with primers that have the restriction sites you want on the 5' end of each primer (i.e. one site per primer).



#3 Clare

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Posted 19 June 2014 - 02:56 AM

Hi,

Thanks for the speedy reply!

I did think about that option however the only enzymes I could use are MauBI and BlpI and by using them I would remove the ZeoR in the vector (but I still have AmpR so should be ok).

I've never used either of these enzymes before...

 

Clare

 

The easiest way to do this is to amplify the new promoter with primers that have the restriction sites you want on the 5' end of each primer (i.e. one site per primer).



#4 phage434

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Posted 19 June 2014 - 07:04 AM

Blunting, cip treatment, and phosphatase are all steps that are difficult to control. In the early 80s this was the only way to do things, so people worked hard to make it work. Now, we have PCR, which makes most of these techniques obsolete. Instead, PCR your vector (most of it, minus the promoter). PCR your insert. Add whatever restriction sites you want to each of the primers. Don't forget 4-6 bases 5' of the restriction site. Use common restriction enzymes. (I've never heard of the ones you mentioned, and probably there is a reason for that). PCR. Purify. Cut. Heat Kill (or purify). Ligate. Transform.



#5 Clare

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Posted 19 June 2014 - 07:38 AM

Hi Phage,

 

I had no idea people routinely PCR the vector as well! I've just always avoided PCR as a general rule for many many years. So you'd even PCR up a 10kb vector? And then sequence the WHOLE thing?

Clare



#6 bob1

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Posted 19 June 2014 - 01:14 PM

10 kb is a big vector, but it is possible to PCR without too much difficulty.  You don't need to sequence the whole vector - just the important bits, which would be resistance genes, promoter regions and maybe the origin of replication site.

 

I don't see why you can't use Xba1 and Not1 as the sites for the new insert to go into - those are the ones you used to remove the old one and you are only blunting, so not adding a bunch of sequence there at all?



#7 phage434

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Posted 19 June 2014 - 04:24 PM

My colleague just did a vector PCR prep with a 12 Kb plasmid. Sequencing the resistance gene is not usually necessary, since only functional plasmids will transform on selective plates. Same for the ori.



#8 Bio-Lad

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Posted 20 June 2014 - 04:33 AM

I agree that PCR amplifying the promoter may be the easiest way to go about this but while you are waiting for your primers to arrive, you may want to try one other thing first.  Since you (hopefully) still have the fragments you digested out, I would suggest trying the blunting with Klenow rather than MBN.  I've tried doing similar ligations before and had quite a bit of trouble getting the MBN to actually do what it was supposed to do and ended up with no colonies as well.  I repeated using Klenow instead and it worked fine the first time around.  Admittedly, it could be something wrong with the way I set up the blunting reactions (can't omit human error) but I've never gotten very good results with it.


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#9 phage434

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Posted 20 June 2014 - 05:19 AM

I agree with Bio Lad, that Klenow would be a much better choice than Mung Bean nuclease. Also, avoid CIP in favor of shrimp alkaline phosphatase or antarctic phosphatase (and use minimal amounts of these, as well).



#10 Clare

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Posted 20 June 2014 - 10:46 AM

Cool.

I can't use XbaI and Not1 as my promoter has both those sites in it (it's actually a 3kb region).

 

10 kb is a big vector, but it is possible to PCR without too much difficulty.  You don't need to sequence the whole vector - just the important bits, which would be resistance genes, promoter regions and maybe the origin of replication site.

 

I don't see why you can't use Xba1 and Not1 as the sites for the new insert to go into - those are the ones you used to remove the old one and you are only blunting, so not adding a bunch of sequence there at all?



#11 Clare

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Posted 20 June 2014 - 10:47 AM

Cheers for the hot tips :) Primers should arrive on Monday.

I agree that PCR amplifying the promoter may be the easiest way to go about this but while you are waiting for your primers to arrive, you may want to try one other thing first.  Since you (hopefully) still have the fragments you digested out, I would suggest trying the blunting with Klenow rather than MBN.  I've tried doing similar ligations before and had quite a bit of trouble getting the MBN to actually do what it was supposed to do and ended up with no colonies as well.  I repeated using Klenow instead and it worked fine the first time around.  Admittedly, it could be something wrong with the way I set up the blunting reactions (can't omit human error) but I've never gotten very good results with it.



#12 Clare

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Posted 20 June 2014 - 10:48 AM

Thanks again! Really appreciate the help guys :D

I agree with Bio Lad, that Klenow would be a much better choice than Mung Bean nuclease. Also, avoid CIP in favor of shrimp alkaline phosphatase or antarctic phosphatase (and use minimal amounts of these, as well).



#13 Clare

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Posted 25 July 2014 - 01:05 AM

Hi!

 

Just wanted to thank you for all your suggestions :D I have clones! wOOt!

 

It seems like we have a contamination in the lab though which is making everyone's cloning a pain in the behind. I'll start a new thread about that.

 

Thanks again!

 

Clare






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