Team:Cambridge/Experiments/Assembly of Reflectin Constructs

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===Gibson Assembly===
===Gibson Assembly===
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We conducted Gibson Assembly of GA1, GA2, GA3 and GA4 constructs according to [Team:Cambridge/Protocols/Gibson_Assembly#Practice | the protocol]]. The volumes of Master Mix and solutions of amplified DNA were the following:
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 +
{|
 +
!GA1
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!GA2
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!GA3
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!GA4
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|-
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|15µl Master Mix
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|15µl Master Mix
 +
|15µl Master Mix
 +
|15µl Master Mix
 +
|-
 +
|2.5µl GA1-1a
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|1.67µl GA2-1a
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|2.5µl GA3-1
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|1.67µl GA4-1a
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|-
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|2.5µl GA1-2
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|1.67µl GA2-2
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|2.5µl GA3-2
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|1.67µl GA4-2
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|-
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|
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|1.67µl GA2-3
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|
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|1.67µl GA4-3
 +
|}
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===Transformation===
===Transformation===
===Results===
===Results===

Revision as of 15:42, 25 August 2011

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OVERVIEW
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This is a placeholder. We should fill it in.

Contents

Construct Design

Primer Design

We should mention expected lengths of products here.

Assembly: first attempt

PCR

In the first round of PCR, we amplified fragments required for the assembly of GA1, GA2, GA3 and GA4 constructs.

Hold 95°C 2 min
Cycling Denaturing 95°C 10 s
Annealing 55°C 20 s
Elongation 72°C 150 s

We decided to use the 55°C annealing temperaure, although the predicted temperature for most primers is 5-10°C higher, because of a low annealing temperature of the VF2 primer.

  • Primers and template DNA provided by our supervisor Paul served as a positive control, but eventually we did not detect any products on the gels.


The pictures below present result of gel electrophoresis of PCR products.

  • In most cases position of a band matches the expected length of DNA fragment. The only exception are GA1-2 and GA3-2 products. According to the position on the gel the length of these DNA fragments is 4-5kb, whereas the predicted length is 3.5kb. Our hypothesis is that we were provided [http://partsregistry.org/Part:pSB1AK3 pSB1AK3] backbone instead of [http://partsregistry.org/Part:pSB1A3 pSB1A3]backbone.
  • For GA1-1, GA2-1, GA3-1 and GA4-1 we obtained two bands: 1000bp and 400bp, with the latter resulting from non-specific priming most probably. We extracted the two bands for GA1-1, GA2-1 and GA4-1 products, labelling the 1000kb and 400bp fragments GAX-1a and GAX1-b respectively.
  • The molecular weight marker that we used in all gels is HyperLadder I, which produces regularly spaced bands ranging from 200 to 10,000bp.

For the gel extraction of DNA we followed the protocol, assuming that one slice of gel is 100μl.

Gibson Assembly

We conducted Gibson Assembly of GA1, GA2, GA3 and GA4 constructs according to [Team:Cambridge/Protocols/Gibson_Assembly#Practice | the protocol]]. The volumes of Master Mix and solutions of amplified DNA were the following:

GA1 GA2 GA3 GA4
15µl Master Mix 15µl Master Mix 15µl Master Mix 15µl Master Mix
2.5µl GA1-1a 1.67µl GA2-1a 2.5µl GA3-1 1.67µl GA4-1a
2.5µl GA1-2 1.67µl GA2-2 2.5µl GA3-2 1.67µl GA4-2
1.67µl GA2-3 1.67µl GA4-3

Transformation

Results

Diagnostics

Assembly: second attempt

PCR

Gibson Assembly

Transformation

Results

What next?


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