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Contents 1 Lab Work - 03/09/09 1.1 Chassis team 1.2 Stochastic switch team 1.2.1 Summary 1.3 Promoter Library Sub-Project 1.3.1 Introduction 1.3.2 PCR Product Cleanup 1.3.3 Conclusion

Lab Work - 03/09/09

Chassis team

Today we continued the lab work on our PCR primers. We again attempted to use two pairs of our primers; the ones to amplify the sleB and cwlJ genes from the B. subtilis genome. We prepared the solutions needed, and followed the PCR protocol. We rehydrated our primers, and diluted them accordingly. The TMs were lowered, following yesterday's failed attempt to 50 and 45 degrees. We prepared four lots of solutions, two with genomic DNA and two without (our controls), containing 2 and 3ul of primer. They were loaded into the PCR machine, which ran for around 2 hours 23 minutes.

When we ran the products on a gel.

Stochastic switch team

Summary

Today we ran the PCR products for sac, ara and sspb on a 0.8% gel with wide wells, which we then cut out using the transilluminator. Goksel also did a ligation of the sspb brick that was purified from the gel using the sigma gel extraction protocol. We ligated the product with the pSB1AT3 plasmid backbone prepared last week. Both the sspb insert and the backbone were cut with EcoRI and SpeI. Today Arun also ran another PCR including our ara region, it worked well so we believe we can use the resulting PCR product for a digest and a ligation.

Promoter Library Sub-Project

Introduction

From the previous lab session the products of the PCR reactions attempting to amplify the sigA promoter library (synthesized sequences of the sigA library containing degeneracies) were run through 1.5% agarose gel in electrophoresis. Analysis of the gel showed that although there were hazy bands forming, these may have not been the result of the PCR products and may have been caused by the primer-dimer complexes formed. The promoter fragments are roughly 100bp however, meaning that there may be little distinction between the promoters and the primer-dimers in 1.5% agarose gel! In light of this experiment, today's task will involve cleaning up the PCR products for another run of DNA gel electrophoresis tomorrow.

PCR Product Cleanup

The PCR Product Kit used to purify the PCR results was the GenElute™ PCR Clean-Up Kit, purchased from Sigma-Aldrich. The protocol used in this experiment is the one given with the kit. There were no changes to the protocol and the steps taken to perform the purification can be seen here:

• Took four Mini-prep Binding Columns and placed them into collection tubes; to each 0.5ml of Column Preparation Solution was added. The tubes were then centrifuged for 1 minute under 12,000g.

• Once the eluate in all of the collection tubes was removed, 200ul of Binding Solution was added to the four PCR Eppendorf tubes containing the PCR DNA (the four tubes containing 40ul of C0, V1, V2 and V3 PCR DNA). These four PCR DNA mixtures were then transferred to the four prepared MiniPrep Binding Columns and centrifuged for 1 minute under 13,000rpm. The eluate was discarded once again with the binding columns returned to their respective collection tubes.

• 0.5ml of Wash Solution was then added to the four binding columns. These binding columns (within the collection tubes) were then centrifuged for 1 minute under 13,000rpm with the resulting eluate discarded. The binding columns were then placed in their original collection tubes and centrifuged again at 13,000rpm; this time with no solutions added.

• The binding columns were then transferred to four new collection tubes (appropriately labelled) whilst the original collection tubes (plus eluate from the 2 minute centrifugation) were discarded. 50ul of Elution Solution was then added to the binding columns and allowed to sit within the tubes on the bench for 1 minute.

• The columns (within their new collection tubes) were then centrifuged for 1 minute under 13,000 rpm but this time it was the eluates which were kept and the binding columns discarded. The four collection tubes containing the eluated PCR products of C0, V1, V2 and V3 PCR reactions were then stored in the -20°C.

Conclusion

With the PCR products purified, another attempt at DNA gel electrophoresis under 1.5% agarose gel should confirm whether the PCR reactions to amplify our synthesized sigA promoter library has worked.