Team:CMUQ/protocols

A1: T7 salt sensor

Introduction

The goal is to amplify T7 salt sensor, digest it along with pSB1C3 plasmid with EcoRI and PstI

Materials

• Polymerase: Fusion Taq or Ampli tas (gold)
• Primers: PrefixF and SuffixR
• Nuclease free H2O
• 5xHF PCR Buffer
• gBlock DNA
• dNTPs
• PCR tubes

Procedure

Preparation of reagents

1. Resuspend gBLOCK in 20µL nuclease free dH2O
2. Resuspend primers to make 100µM stock then dilute to make a 10µM stock
3. Add the following reagents to a PCR tube:

4. Run the PCR machine

5. Digest amplified sequence with EcoRI and PstI
6. Digest pSB1C3 vector with EcoRI and PstI
7. If you clone into a vector that expresses a fluorescent protein, other than red since you are cloning in red, then colonies that still express that fluorescent protein will be negative clones and the red or white colonies will be the ones that you want.
8. Run on an agarose gel, cut out the bands and purify.
9. Send clones for sequencing