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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 Primers 1.1.3 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 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.

Preparing the PCR

The table below shows the amounts of substances added to each PCR reaction:

PCR Reaction Table

No.	Water (ul)	PCR Mix (ul)	Forward Primer (ul)	Reverse Primer (ul)	Template (ul)	Polymerase used (ul)	Total Volume (ul)
1	31	15	1	1	200uM (1ul)	1	50
2	31	15	1	1	200uM (1ul)	1	50
3	31	15	1	1	200uM (1ul)	1	50
4	31	15	1	1	200uM (1ul)	1	50