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
- - - - -

How to ligate ds-oligos into a vector?


  • This topic is locked This topic is locked
23 replies to this topic

#1 kroger

kroger

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 25 August 2004 - 11:04 AM

Hi,guys:
I try to ligase a ~40bps DNA into a single digested Vector.I got this ~40 bps DNA from annealing two complementary oligos, there are two same restriction enzyme sites in both sides of oligos. I didn't phosphorylate my oligos and also didn't dephosphorylate my vector.I use the big molar ration of insert DNA to digested Vector,try to kill reaction. But I didn't get any ligation product, only this digested vector ligase itself. some person has such experience not to phosphorylate and dephosphorylate vector,and got product,but I didn't succeed. do you have some suggestions? or I must do phorsphorylation and diphosphorylation?
appreciate your help!
Viki

#2 lyrezxl

lyrezxl

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 25 August 2004 - 05:41 PM

hi, :rolleyes:
if i was u, i'll do phorsphorylation and diphosphorylation.
it's easy, reliable, and always save ur time and pain. :lol:
cheers!
lyre

#3 bitpas

bitpas

    member

  • Active Members
  • Pip
  • 18 posts
0
Neutral

Posted 26 August 2004 - 01:44 PM

definitley do dephosphoylation of your vektor or you'll be screenin' colonies for the rest of your life.. :D

greetings!

#4 kroger

kroger

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 26 August 2004 - 02:40 PM

definitley do dephosphoylation of your vektor or you'll be screenin' colonies for the rest of your life..  :D 

greetings!

thank you very much,so I also need to phosphorylate my oligos?

#5 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 27 August 2004 - 02:49 AM

yes, you definitely need to phoshorylate your oligos.

PNK + ATP will do the job.

But, if you can afford it, its more reliable to order your oligos phophorylated already.

mike
--- He who finds typos may keep them! ---

#6 phegene

phegene

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 30 August 2004 - 06:11 AM

If you are still having trouble, gel purify your oligos first on a polyacrylamide gel, then anneal the two oligos and phosphorylate them. Dephosphorylate your digested vector and gel extract that as well. Then set up your ligation in 10uL. I've done this many times and it will even work with the quick ligation kits. Good luck.

#7 kroger

kroger

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 30 August 2004 - 08:56 AM

If you are still having trouble, gel purify your oligos first on a polyacrylamide gel, then anneal the two oligos and phosphorylate them.  Dephosphorylate your digested vector and gel extract that as well.  Then set up your ligation in 10uL.  I've done this many times and it will even work with the quick ligation kits.  Good luck.

thank you very much, why do you purify the oligos first?
:)

#8 phegene

phegene

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 01 September 2004 - 09:13 AM

When ordering PCR primers or sequencing primers, typically around 20bases, it is not necessary to gel extract. When longer oligos are ordered for cloning, it is very important to have the exact sequence ordered. Since no companies can guarantee 100% efficiency in the synthesis, there will be a certain percentage of the Oligos received that is not full-lenght product. This amount increases exponentially as the oligo length increases. Gel extracting the oligos reduces the amount of shorter oligo product in your sample and thus increases the chances for a successful ligation.
I have worked with 60-90 base oligos and I always see a large amount of shorter products. If I do not gel extract first, I have never been able to clone the annealed oligos.
Good luck!

#9 tfitzwater

tfitzwater

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 96 posts
3
Neutral

Posted 01 September 2004 - 01:56 PM

One potential problem with annealing two primers that contain restriction sites is that they also tend to ligate to themselves due to the inverted repeat nature of many restriction recognition sites.

Plug the sequences into the OligoAnalyzer program at the www.idtdna.com site and check for hairpins, homo-dimers and hetero-dimers.

For a 40 bp insert, phosphorylated insert:dephosphorylated vector ratios of 10:1 and 15:1 should be sufficient.

#10 kroger

kroger

    member

  • Active Members
  • Pip
  • 20 posts
0
Neutral

Posted 05 September 2004 - 06:06 PM

When ordering PCR primers or sequencing primers, typically around 20bases, it is not necessary to gel extract.  When longer oligos are ordered for cloning, it is very important to have the exact sequence ordered.  Since no companies can guarantee 100% efficiency in the synthesis, there will be a certain percentage of the Oligos received that is not full-lenght product.  This amount increases exponentially as the oligo length increases.  Gel extracting the oligos reduces the amount of shorter oligo product in your sample and thus increases the chances for a successful ligation. 
I have worked with 60-90 base oligos and I always see a large amount of shorter products.  If I do not gel extract first, I have never been able to clone the annealed oligos. 
Good luck!

thank you very much.
If I use polyacrylamide gel to isolate DNA,is the same protocol as isolating protein by polyacrylamide gel?

#11 jadefalcon

jadefalcon

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 223 posts
1
Neutral

Posted 06 September 2004 - 03:41 AM

I'm not quite sure about your protein isolation, but there are commercial kits (e.g. Qiagen) that have protocols for DNA Extraction out of agarose and PA gels.

mike
--- He who finds typos may keep them! ---

#12 5900P1

5900P1

    member

  • Members
  • Pip
  • 2 posts
0
Neutral

Posted 21 September 2004 - 04:45 PM

DNA in PAGE can by eluted into TE and 0.1%SDS overnight OR you can put it into dialysis tubing and eletroelute it.

#13 nanaz

nanaz

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 07 January 2005 - 09:11 AM

Hi
I read your replies to enthusiast and I had similar problems and my primers also have the restriction sites at the end of them of the same enzyme to be later ligated in the sinly cut vector. I tried dephos my vector and ordered my primers 5' phosphorylated. I annealed the primers by heating 94 C fro 1 min and at room temp for 1hr. and also as suggested by some one I annealed them by placing in a water bath 70C for 1-2hr and bringing temp down slowly for a whole day say 10 each hr. I still get as many colonies on my control plate (no insert) after ligation. I even tried cutting the dephosphorylated digested vector off the gel and purify it. I did however never gel purify my insert (the annealed!! primers). How would I do that? Do I just let anneal and run on gel and purify or......???
I would be grateful if you could suggest why I am getting similar no. of colonies on my control (no insert) plate as my ligation plates?
Thank you in advance

#14 phegene

phegene

    member

  • Active Members
  • Pip
  • 12 posts
0
Neutral

Posted 07 January 2005 - 10:12 AM

Depending on the size of your oligos, you probably need to purify them. If they are under 30 bases, make sure to anneal properly (see below, just skip purification, add together ~800pmoles of each, anneal in 100uL, dilute as described and use in ligation). Since you are getting similar colonies on your no insert plate, your vector is not completely cut....Use more enzyme in a new reaction, and CIP for 1 hour. You can ethanol precipitate or gel extract before using in the ligation. Here are some protocols:

To purify single stranded oligos, you must run them on a 10% TBE-Polyacrylamide gel. Purify 900pmoles of primer (for ~45 bases) and mix with RNA dye (formamide based dye). Heat 2min at 90C, then load on gel. Run at 30mA until dye is halfway down. To see the oligo, you will need a fluor-coated silica plate (Ambion #10110, Fisher also sells larger sizes and amounts). Cover your silica plate with saran wrap, place your gel on it. You can view the oligos by "UV shadowing": hold the plate at a 45 degree angle above a UV Box, a purple band will be visible. The darker band near the top, if multiple bands are seen, should be your correctly sized DNA oligo. cut it out with scalpel and place in 1.5mL tube, you can break the gel piece at this point, it will help with the elution. Elute in elution buffer at RT overnight, or at 65C for 2 hours. After the elution, spin down your tube, remove the supernate and save to a new tube. Add 200uL buffer to the gel piece. Vortex and spin down, combine with the first 200uL. Ethanol precipitate with 1uL of 10% Dextran Blue to view the DNA after spinning it down. Resuspend each oligo in 10uL.
Elution Buffer: 600uL of 5M AmAc, 60uL 1M MgAc, 120uL 0.5M EDTA, 60uL 10%SDS, 5.16mL H2O.

To Anneal the purified oligos: Combine plus and minus oligos, total of 20uL. Add 10uL of Promega Buffer E and 70uL H20. Place in boiling water bath for 5min. Remove the water bath from the heat source and let cool to RT. (I use a beaker on a hot plate, and remove it from the hot plate after 5 min and leave the samples in it white it cools down.)
Take 10uL of the annealed oligos, dilute in 990uL H2O. Use 2uL of this dilution for ligation.


If you did not order your oligos with a 5' phosphate, do the following after annealing:

2uL annealed oligos, 1uL 10mM ATP, 0.5uL 10XPNK Buffer, 0.5uL H20, 1uL PNK
1hour at 37C, then heat inactivate by adding 0.5uL 0.5M EDTA, 10min 68C. Dilute to 200uL. Use 2uL of this dilution for your ligations.

This protocol was derived from Methods in Enzymology Vol 346, Designing and Characterizing Hammerhead ribozymes for Use in AAV Vector-Mediated retinal gene therapies, by Fritz et al, 2002. pp358-377.

#15 nanaz

nanaz

    member

  • Active Members
  • Pip
  • 6 posts
0
Neutral

Posted 10 January 2005 - 05:08 AM

To Anneal the purified oligos: Combine plus and minus oligos, total of 20uL. Add 10uL of Promega Buffer E and 70uL H20. Place in boiling water bath for 5min. Remove the water bath from the heat source and let cool to RT. (I use a beaker on a hot plate, and remove it from the hot plate after 5 min and leave the samples in it white it cools down.)
Take 10uL of the annealed oligos, dilute in 990uL H2O. Use 2uL of this dilution for ligation.


my total primer size is 37 bp and so I guess I can skip the gel purification of the primers or can I do it using say Qiagen gel extraction kit?
also I have never used buffer E so I searched Promega and could only find buffer A, B, C and D for double digestion of DNA!!
when you say water bath do uou mean 70C
I have tried doing the technique you suggest but without the buffer E and putting the primers to anneal in a water bath bringing down temp by 10 degrees carrying on for the whole day long.
and at the end I got the colonies I said before!!
Thanks again




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

©1999-2013 Protocol Online, All rights reserved.