Team:Newcastle/Labwork/1 September 2009

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(→Preparing the PCR)
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Today's work involves the amplification of the promoter library (a collection of ''sigA'' promoters specified by degenerate sequences) using PCR.
Today's work involves the amplification of the promoter library (a collection of ''sigA'' promoters specified by degenerate sequences) using PCR.
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===Preparing the PCR===
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===Preparing the Primers===
The first task carried out was the rehydration of the primers involved with this sub-project. A final concentration of 200uM is the desired value and to get this for each primer, the following calculation was observed:
The first task carried out was the rehydration of the primers involved with this sub-project. A final concentration of 200uM is the desired value and to get this for each primer, the following calculation was observed:
Amount of water to be added (ul) = amount of primer (nMoles) x 5
Amount of water to be added (ul) = amount of primer (nMoles) x 5
-
The table below shows the oligos involved in this practical along with the primer amounts (in nMoles) and the amount of water needed (in ul) to make the primers at a concentration of 200uM
+
The table below shows the oligos involved in this practical along with the primer amounts (in nMoles) and the amount of PCR water needed (in ul) to make the primers at a concentration of 200uM
   
   
{| border="1"
{| border="1"
|+ A table showing the oligos with the amount of water needed
|+ A table showing the oligos with the amount of water needed
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! Oligo !! Purpose !! Amount (nMoles)!! Water added (ul)
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! Oligo !! Purpose !! Amount (nMoles)!! PCR Water added (ul)
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! IDT 60396420
! IDT 60396420
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All of these oligos were then aliquoted and further diluted 1:100 with PCR water and stored in the -20C freezer. The primer stocks are kept in Neil's yellow box whereas the dilutions in enzymes are kept in the a separate box.
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Revision as of 08:56, 2 September 2009

Contents

Lab Session 01/09/2009

Promoter Library Sub-Project

Introduction

Up until now, this sub-project has seen the pGFP-rrnB plasmid backbone digested and linearised with restriction enzymes EcoRI and NheI. The resulting 8kb fragment (which is the plasmid backbone) has been successfully excised from agarose gel and purified; it is into this plasmid backbone in which we will place our sigA promoters.

Today's work involves the amplification of the promoter library (a collection of sigA promoters specified by degenerate sequences) using PCR.

Preparing the Primers

The first task carried out was the rehydration of the primers involved with this sub-project. A final concentration of 200uM is the desired value and to get this for each primer, the following calculation was observed:

Amount of water to be added (ul) = amount of primer (nMoles) x 5

The table below shows the oligos involved in this practical along with the primer amounts (in nMoles) and the amount of PCR water needed (in ul) to make the primers at a concentration of 200uM

A table showing the oligos with the amount of water needed
Oligo Purpose Amount (nMoles) PCR Water added (ul)
IDT 60396420 Forward Primer 19.6 98.0
IDT 60396419 Reverse Primer 21.9 109.5
alta D68423AW Control 0.06 300
alta D68425AW Variant 2 (V2) 0.06 300
alta D68424AW Variant 3 (V3) 0.06 300
alta D68426AW Variant 1 (V1) 0.06 300
alta D68427AW 19mer Primer 0.08 400

All of these oligos were then aliquoted and further diluted 1:100 with PCR water and stored in the -20C freezer. The primer stocks are kept in Neil's yellow box whereas the dilutions in enzymes are kept in the a separate box.



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