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Basic Gateway Cloning

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Biological and Medical Sciences > Classification by Description > Reagents, Kits and Assays > DNA and RNA > Cloning


Branch: Protocol-Wiki
Type: Protocol
Intention: For Publication
Read/Write Permissions: Open Access
Authors :
27.06.2016 - Christopher Dyer  -  Sci-Mate   
13.12.2010 - John Stewart   


This very simple protocol describes how to move a sequence or gene of interest into or between Gateway vectors.

The differences between Gateway cloning and traditional cloning methods are that digestion and ligation occurs within the same tube, and it works virtually every time.

To use this protocol, your sequence or gene of interest must be flanked by the relevant Gateway restriction sites (see also Designing a Gateway Cloning Strategy). If the sequence or gene of interest is NOT already surrounded by the needed sequences, this can be added by PCRing your sequence using primers with Gateway restriction sites.

Contents

[edit] Step-by-step Protocol for Gateway cloning

  1. Prepare the "Gateway Cloning Reaction (Digestion and Ligation)" as described in the table below and leave for 1 hour at 25oC.
  2. Add 2ul of 2ug/ul Proteinase K solution and leave for 10 minutes at 37oC.
  3. Transform bacteria and select with appropriate antibiotics (see notes below).
  4. Mini-, Midi-, or Maxi-prep the DNA for restriction or sequence analysis.

[edit] Gateway Cloning Reaction (Digestion and Ligation)

The following solutions should be prepared from reagents that have been thawed on ice.


Ligation
(ul)
Positive Control
(ul)***

Negative Control
(ul)***

DNA Fragment (20-50 fmol*)
or
Expression vector (150 ng)
or
Entry vector (150 ng)
1-7

1-7
Donor vector (150 ng)
or
Destination vector (150 ng)
1
1
1
pEXP7-tet (as supplied)

2

TE
0-6
5
2-8

BP Clonase II**
or
LR Clonase II**

2
2

 * To convert femtomoles of DNA to nanograms, use the following forumula:  Z = (Y x N x 66/10^5), where Z is the quantity of DNA in ng; Y is the quantity of DNA in femtomoles and N is the number of base pairs.

 ** Thaw on ice, and vortex before use. BP Clonase II should be used to recombine Gatewayed PCR fragments or Expression vectors, which have aatB restriction sites, with Donor vectors, which have aatP restriction sites; and LR clonase II in reactions to recombine Entry vectors, which have aatL restriction sites, with Destination vectors, which have aatR restriction sites (see Gateway Lambda Phage Restriction Sites).

 *** This reaction works in most cases with such efficiency that controls may be ommited until a problem with the outcome is recognized.

[edit] Transfection, Selection and Analysis of DNA

Use 1 ul of supernatant from the Cloning Reaction to transfect any recA endA E. coli strain (using any standard transfection protocol).

Following transfection, bacteria should be plated on agar plates containing antibiotic corresponding to the resistance cassette of the desired vector, and left to grow overnight.

  • Donor and Entry vectors generally confer Kanamyacin resistance, while
  • Destination and Expression vectors vary (refer to the vector map).

A successful Gateway cloning reaction should yield between 100 and 2 000 colonies.

The efficiency of this reaction is such that it should not be necessary to mini-prep more than 4 colonies. With confidence, it is even possible to go straight to midi-, or maxi-prep for restriction enzyme digest or sequencing.

[edit] Note

This is a basic protocol intended to simplify what is an very easy technique to perform. However, the technology behind Gateway is complex and fascinating. If more experienced users would like to add information, they are more than welcome to edit or discuss this article, or create spin off Articles with more 'high end' details.





  • This page was last modified 10:44, 27 June 2016.
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