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Lab Work - 29/07/09

Introduction

In yesterday's lab session we conducted mini-preps for three sets of JM109 E. coli cells which contained BioBricks BBa_C0056, BBa_B1002 and BBa_R0077 (seen as we picked three colonies for each of the BioBrick trasnformants there was a total of nine mini-preps conducted). A sample of these prepped plasmids were then run on agarose gel; although the undigested plasmid samples couldn't determine whether the DNA was in fact the BioBrick incorporated within the correct plasmid the gel certainly showed that the samples were consistent (trulin out contamination).

The next step in the lab plan is to digest samples of these 9 mini-preps and then run these fragments on agarose gel by DNA Gel Electrophoresis. As well as this, the team needs to consider another attempt at transforming E. coli with the BioBricks BBa_C0077 and BBa_C0076 (the transformations which didn't yield colonies on LB + ampicillin plates). In addition the team needs to start to freeze down cells which will probably be needed in the future.

Practical Outline

Before the day is out the team needs to carry out the following:

1. Carry out a restriction digest on the 9 mini-preps using enzymes EcoRI and PstI
2. Run the digest products on gel through DNA Gel Electrophoresis.
3. Transform JM109 E. coli cells with BioBricks BBa_C0077 and BBa_C0076
4. Inoculate tubes containing 5ml LB (+ antibiotic) with the cells that are to be frozen down

Procedure

Restriction Digests

The restriction enzyme digests were conducted by firstly inserting the following components to Eppendorf tubes:

The solutions involved in the Restriction Enzyme digests

	BBa_C0056	BBa_B1002	BBa_R0077
DNA (ul)	1	10	10
Water (ul)	15	6	6
Buffer (ul)	2	2	2
EcoRI (ul)	1	1	1
PstI (ul)	1	1	1

A couple of points to make:

1. Three colonies were taken from each plate containing BioBrick transformants so there is a total of 9 mini-prep digests - 3 BBa_C0056 digests, 3 BBa_B1002 digests and 3 BBa_R0077 plasmid digests.
2. It was decided that only 1ul of BBa_C0056 would be digested (as opposed to 10ul of DNA) - this is because the gel produced the previous day showed that the DNA concentration of BBa_C0056 mini-prep was really high.

The substances were added in the following order: 1) DNA, 2)Water, 3)Buffer, 4)Enzyme

Because the components of the digest were pipetted against the sides of the 9 Eppendorf tubes, the tubes were spun down for a pulse to mix the contents together. The 9 Eppendorf tubes were then placed in the 37C water bath for 1 hour.

DNA Gel Electrophoresis

The DNA samples were the prepared in the way suggested by the DNA Gel Electrophoresis protocol (i.e. 9ul of restriction digest solution and 1ul of loading buffer/dye) and loaded into the 0.8% agarose gel (along with the HindIII DNA ladder). DNA gel electrophoresis was allowed to commence for 40 minutes under 100 volts. Once this procedure was accomplished, the gel was analysed under UV light by GelDoc. The photograph can be seen under the 'Results' section.

Transforming JM109 cells with BBa_C0077 and BBa_C0076

The team are going to reattempt the transformations of JM109 cells with BBa_C0077 and BBa_C0076 - this time, we will plate the transformants on LB + kanamycin plates and NOT LB + ampicillin!. Again the team followed the [http://www.promega.com/tbs/tb095/tb095.html protocol set by Promega] and only made a few changes:

• Step 3 - instead of adding 100ul of Competent Cells to the culture tubes, only 50ul was transferred.
• Step 4 - the DNA added to the competent cells was 1ul instead of 10ul (this is because we only had approximately 2-3ul of DNA remaining!)
• Step 8 - the media used to grow the transformant cells was LB and not SOC

Once the transformation process was completed, both sets of E. coli cells (containing BBa_C0077 and BBa_C0076) were plated out on kanamycin LB plates. These plates were then transferred to the 37C incubator for overnight growth.

Preparing cells for freezing down

The cells to be frozen down included the following: E. coli containing RFP, E. coli containing GFP, JM109 E. coli containing BBa_C0056, JM109 E. coli containing BBa_B1002, JM109 E. coli cells containing BBa_R0077 and E. coli cells containing pMUTIN4 plasmid.

6 tubes were labelled and to each of them, 5ml of LB was added. Anti-biotics were then added to the appropriate tubes (25ul of each antibiotic is the volume used for 5ml of LB) and the colonies added to the right tubes. The tubes were laid out as follows:

Contents of tubes

Tube Number	Cells to be frozen down	LB (ul)	Antibiotic 1	Antibiotic 2
1	E. coli + RFP	6	Ampicillin (0.025ul)	-
Water (ul)	15	6	6
Buffer (ul)	2	2	2
EcoRI (ul)	1	1	1
PstI (ul)	1	1	1

Results

These are the wells:

• Lane 1 = blank
• Lane 2 = HindIII DNA ladder
• Lane 3 = BBa_C0056 mini-prep (from colony 1) digest
• Lane 4 = BBa_C0056 mini-prep (from colony 2) digest
• Lane 5 = BBa_C0056 mini-prep (from colony 3) digest
• Lane 6 = BBa_B1002 mini-prep (from colony 1) digest
• Lane 7 = BBa_B1002 mini-prep (from colony 2) digest
• Lane 8 = BBa_B1002 mini-prep (from colony 3) digest
• Lane 9 = BBa_R0077 mini-prep (from colony 1) digest
• Lane 10 = BBa_R0077 mini-prep (from colony 2) digest
• Lane 11 = BBa_R0077 mini-prep (from colony 3) digest
• Lane 12 = HindIII DNA ladder