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Lab Work - 08/09/09

Metal Sensing Team

Introduction

Yesterday's lab session saw the Metal Sensing team innoculate 6 tubes of 5ml LB (plus ampicillin) with six cultures of DH5-alpha transformed with BBa_J33206 in plasmid pSB1A2 in the morning. By the time afternoon had arrived, the bacteria had grown to sufficient numbers; three of these cultures were then used to further inoculate 3 flasks of 50ml LB+amp. The three flasks of 50ml LB+amp + transformant E. coli cells were then placed in the orbital incubator and will take part in today's midi-prep. Meanwhile the six tubes were used in a mini-prep attempt but alas, the procedure went wrong and had to be abandoned.

Today's lab session will focus on creating midi-prepped plasmids from the inoculated 50ml LB+amp flasks as well as carry out restriction enzyme digest analysis on a sample. The samples will then be run on agarose gel by DNA gel electrophoresis.

Procedure

Midi-prep of transformant E. coli cells

To carry out the midi-prep we used Sigma-Aldrich's GenElute midi-prep kit and with it, the protocol. There were no changes to the protocol except for an additional step after step 10b - an additional 2 minute spin to ensure ethanol removal. The route we took was the Spin method.

The only thing to note is that we did not midi-prep all of the samples placed in the flask; cultures 1 and 2 (both deriving from the 200ul LB+amp plates) were both spun down with supernatants removed (culture 2 from the 500ul plate was discarded) with culture 1 being placed in the freezer for storage and culture 2 used for the midi-prep process. Here's a summary of the process:

• 50ml of culture 1 was spun down for 10 minutes at 5,000g. The supernatant was aspirated off and the pellet was resuspended in 4ml Resuspension/RNAse solution.

• 4ml of lysis solution was immediately applied and after inverting the falcon tube 6 times, the solution was allowed to sit on the bench for 4 minutes. 4ml neutralisation solution was added once the time had expired and the tube inverted 6 times again.

• 3ml of binding solution was added and after 2 inversions the solution was placed into the filter syringe. With the plunger removed from the syringe, the lysate remained in the syringe for 5 minutes. During this time, 4ml of Column Preparation Solution was allowed to be run through the midiprep binding column (by centrifugation).

• The plunger was then applied to the syringe and half of the lysate (which had been sitting in the syringe barrel for 5 minutes) was injected into the midiprep binding column. The binding column was placed in the centrifuge at 3,000g for 2 minutes and once the eluate had filtered through the remaining half of the lysate was added and treated in the same way.

• 4ml of Wash Solution 1 was then added to the binding column and centrifuged for 2 minutes before 4ml of Wash Solution 2 was added and then placed in the centrifuge for 2 minutes again. The eluate was discarded before the tubes were placed under a further 2 minute spin to ensure all of the ethanol had gone.

• 1 ml of Elution solution was then added to the midiprep binding column before the column was spun for 5 minutes at 3,000g. The eluate was then harvested at the bottom of the binding column and transferred to the freezer for storage.

Restriction Enzyme digests

Samples were taken from the midi-prepped plasmid from culture 2 (from 200ul plate) and placed into 4 Eppendorf tubes with the following contents:

• Eppendorf tube 1 = pSB1A2 with BBa_J332306 + NO ENZYMES
• Eppendorf tube 2 = pSB1A2 with BBa_J332306 + EcoRI
• Eppendorf tube 3 = pSB1A2 with BBa_J332306 + PstI
• Eppendorf tube 4 = pSB1A2 with BBa_J332306 + EcoRI + PstI

The table below shows the contents of each Eppendorf tube:

Contents of the four samples of BBa_J33206 in pSB1A2 plasmid

	pSB1A2 alone	pSB1A2 + EcoRI	pSB1A2 + PstI	pSB1A2 + EcoRI + PstI
DNA (ul)	2	2	2	2
Restriction Buffer (ul)	2	2	2	2
Restriction Enzyme(ul)	0	1	1	2
Distilled water(ul)	16	15	15	14

The digests were allowed to progress for 1 hour in the waterbath, which was set to 37C.

DNA gel electrophoresis analysis

We loaded the gel in the same way suggested by the DNA gel electrophoresis protocol except we loaded 6ul of DNA samples (5ul of DNA and 1ul of loading buffer/dye). After about 30 minutes of electrophoresis under 90v, the gel was taken to the dark room and analysed using GelDoc. The photograph can be seen in the 'Results' section below.

Results

The two gel photographs above show the restriction enzyme digests of the pSB1A2 plasmid, which contains the BBa_J33206 BioBrick.