Team:Cambridge-JIC/Protocol

From 2014.igem.org

(Difference between revisions)
Line 15: Line 15:
</ol>
</ol>
 +
<h3>PCR</h3>
 +
<p>
 +
In this step we will amplify the fragments required for our plasmids with
 +
Phusion DNA polymerase. PCR will amplify a fragment from a DNA
 +
template, with the help of short DNA sequences complementary to the
 +
template that demarcate the ends of the fragment. In our case, the
 +
template will be a similar plasmid with RFP-LTI in place of our GOI.
 +
</p>
 +
!!Put in picture of the plasmid !!
 +
 +
<p>
 +
The plasmid backbone will be split into 3 pieces, as it is quicker and less
 +
error prone to PCR short fragments (<5kb). The fragments will be
 +
amplified with the following pairs of single stranded DNA primers
 +
(length of the fragments in brackets):
 +
</p>
 +
 +
<ol>
 +
<li> nosT_F and P2_B (2137bp) </li>
 +
<li> P2_F and P1_B (2501bp) </li>
 +
<li> P1_F and 35s_B (3000bp) </li>
 +
</ol>
 +
 +
<p>
 +
A fourth fragment will be our GOI
 +
All the fragments are designed to overlap with each other by 20-40 bp for
 +
the subsequent Gibson assembly reaction.
 +
</p>
</html>
</html>

Revision as of 09:28, 21 July 2014

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General outline of the method:

  1. Polymerase Chain Reaction (PCR) to obtain DNA fragments
  2. Gel electrophoresis to select the correct fragments
  3. DNA purification for plasmid assembly
  4. Gibson Assembly
  5. E. coli transformation and plating
  6. Colony analysis and selection by colony PCR
  7. Miniprep and sequencing

PCR

In this step we will amplify the fragments required for our plasmids with Phusion DNA polymerase. PCR will amplify a fragment from a DNA template, with the help of short DNA sequences complementary to the template that demarcate the ends of the fragment. In our case, the template will be a similar plasmid with RFP-LTI in place of our GOI.

!!Put in picture of the plasmid !!

The plasmid backbone will be split into 3 pieces, as it is quicker and less error prone to PCR short fragments (<5kb). The fragments will be amplified with the following pairs of single stranded DNA primers (length of the fragments in brackets):

  1. nosT_F and P2_B (2137bp)
  2. P2_F and P1_B (2501bp)
  3. P1_F and 35s_B (3000bp)

A fourth fragment will be our GOI All the fragments are designed to overlap with each other by 20-40 bp for the subsequent Gibson assembly reaction.

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