Corum:T4 Ligation: Difference between revisions

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For a 10 μL ligation reaction:
For a 10 μL ligation reaction:


* 7 μL linker dsDNA (either 100nM standard concentration linker hybridization product or standard digested and purified PCR product, which is usually ~100nM)
* 3 μL sterile ddH<sub>2</sub>O
* 0.5 μL backbone dsDNA (usually ~10 nM) (Note: ratio of backbone to linker should be 10-20:1. The specific amounts of linker and backbone dsDNAs may need to be adjusted to keep this ratio for non-standard concentrations)
* 1 μL 10X ligation buffer
* 1 μL 10X ligation buffer
* 1 μL 10mg/ml BSA
* 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
* 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
* 0.5 μL T4 ligase
* 0.5 μL T4 ligase
(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)


==Procedure==
==Procedure==


# Add the reaction components together in order listed above. Mix gently and spin.
# Add the reaction components together in order listed above. Mix gently and spin.
# Include -linker control reaction (sub sterile ddH<sub>2</sub>O for linker).
# Incubate 16 °C overnight (8 hr minimum).
# Incubate 16 °C overnight (8 hr minimum).
# [http://openwetware.org/wiki/Corum:Transformation Transform] 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.


==Notes==
==Notes==
Line 27: Line 30:


Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-------------
# *'''SC 18:03, 30 August 2012 (EDT)''': Removed 1mg/ml BSA component from protocol, per the fact that it is not recommended in the [http://www.neb.com/nebecomm/products/productm0202.asp NEB protocol].


==References==
==References==
'''Relevant papers, books, and websites'''
<!-- If this protocol has papers or books associated with it, list those references here.-->
<!-- Try the [[Template:FormatRef|FormatRef template]]-->
#[http://www.neb.com New England Biolabs]
#[http://www.neb.com New England Biolabs]


Latest revision as of 15:05, 30 August 2012

  • SC 15:16, 19 June 2012 (EDT):

Overview

Standard DNA ligation using NEB T4 ligase to form a vector.

Materials

For a 10 μL ligation reaction:

  • 3 μL sterile ddH2O
  • 1 μL 10X ligation buffer
  • 5 μL ~100nM linker dsDNA (either hybridization product or digested, purified PCR product)
  • 0.5 μL ~10nM backbone dsDNA (gel extracted digestion product from donor plasmid)
  • 0.5 μL T4 ligase

(Note: ratio of backbone to linker given here is ~1:100. This may need to be adjusted in some cases.)

Procedure

  1. Add the reaction components together in order listed above. Mix gently and spin.
  2. Include -linker control reaction (sub sterile ddH2O for linker).
  3. Incubate 16 °C overnight (8 hr minimum).
  4. Transform 2.5 μL rea(or more, if necessary, to obtain an acceptable amount of colonies) into chemically competent cells.

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
  3. Anecdotal observations that might be of use to others can also be posted here.

Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.

  1. *SC 18:03, 30 August 2012 (EDT): Removed 1mg/ml BSA component from protocol, per the fact that it is not recommended in the NEB protocol.

References

  1. New England Biolabs

Contact

or instead, discuss this protocol.

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