Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

Changing the vector

cloning

55 replies to this topic

#1 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 05 August 2014 - 12:22 AM

Hi,

I need an advice where to start and what point need to be taken in advice. I have a gene that needs to be moved from vector A and inserted in vector B. 

there is no similarity in the cloning site in terms of restriction enzymes.

Thank you in advance,

 



#2 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,737 posts
400
Excellent

Posted 05 August 2014 - 02:54 AM

Amplify the gene with primers that contain the restriction sites you want to use.



#3 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 05 August 2014 - 04:12 AM

It is 4kb gene. Do you think I can do that by Primer amplification ?

#4 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,475 posts
251
Excellent

Posted 05 August 2014 - 05:32 AM

Yes. This should be routine, with sufficient extension time.



#5 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,737 posts
400
Excellent

Posted 05 August 2014 - 02:58 PM

Make sure the restriction sites are at the 5' end of the primers. Also ensure that you have 6 bp (any sequence will do) 5' of this, so as to give the RE something to bind to.

 

Primers should look like this:

 

6bp<REsite>Specific sequence



#6 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 05 August 2014 - 06:56 PM

Ok! Thank you very much bob. I will be inserting This gene into pTRE3G vector . According to the MCS I can use 11 restriction enzymes. Is there is any preference? What is the most easier tool/ program to use to design the primers? (I am mac user)



#7 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,475 posts
251
Excellent

Posted 05 August 2014 - 07:30 PM

Use enzymes you've heard of before and that you already have in your freezer. Choose ones that can be heat killed to save you a purification step after cutting. EcoRI, PstI, XhoI, HindIII, XbaI, KpnI



#8 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 05 August 2014 - 08:02 PM

Thank you indeed, but non of these enzymes in MCS I will google for my options. What about primer design tool ? How long the minimum sequence overlap between the primers and the gene that need to amplify ?



#9 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,737 posts
400
Excellent

Posted 05 August 2014 - 08:44 PM

I would go for BamHI and BglII, neither can be heat inactivated so you will have to clean up after the reaction, but they should work in the same buffer (at least NEB tells me so) so a double digest is a possibility.



#10 Bio-Lad

Bio-Lad

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 85 posts
16
Good

Posted 10 August 2014 - 07:29 PM

I'm a bit late to this but for primer designing, Primer3 works pretty well.(


Try pLOT, the a free plasmid mapping tool!

http://plasmidplotter.blogspot.com/


#11 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 10 August 2014 - 11:47 PM

thanks you Bip-Lab! Can you advice me what is the minimum sequence overlap between the primers and the gene that need to amplify



#12 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,737 posts
400
Excellent

Posted 11 August 2014 - 12:11 AM

Standard primers of 20-25 bases will work fine for the coding sequence. The size of the things you add don't matter for the amplification, only the part that is specific, so only base Tm calculations off the specific part of your primers.

 

Note that if you are trying to get the whole coding sequence for expression, you don't have much option as to where to place the primers (from the ATG and stop codons respectively), its just a matter of adding or subtracting a base or two to get the Tm about the same for each.



#13 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 11 August 2014 - 12:15 AM

thank you! yes exactly I am trying to get the whole coding sequence. it is it 4Kb. then later I will legate it to 3.4 vector!



#14 bob1

bob1

    Thelymitra pulchella

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 5,737 posts
400
Excellent

Posted 11 August 2014 - 01:28 PM

Ok in that case the specific part of your primers with start with ATG for the forward and for the reverse it will be the reverse complement of the stop codon.

 

Note that if you are trying to tag the gene then you need to make sure that the tag is in-frame with the insert.



#15 RNA woman

RNA woman

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 44 posts
0
Neutral

Posted 11 August 2014 - 05:52 PM

Thank you bob1 and everyone respond to mu questions. Actually I will not tag the gene. I have more one more questions do I need to linearise the the vector before cloning? 

Also if anybody can refer me to an useful site to get idea how to make point mutation starting from GOI in a vector. I am bio-physicist and really new to those things. 







Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.