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Contents 1 Lab Session 01/09/2009 1.1 Promoter Library Sub-Project 1.1.1 Introduction 1.1.2 Preparing the PCR

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 PCR

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

A table showing the oligos with the amount of water needed

Oligo	Purpose	Amount (nMoles)	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