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Formal Lab Session - 18th September 2009

Stochastic Switch Team

Today we did midipreps of the ara transformation and digested 10ul to run on a gel (EcoRI + PstI) Two bands could be seen one for the backbone and one correctly sied for the insert (~200bp) so this midiprep can be taken forward in order to do the ara-sspb double clone.

Today we also made up some fresh DNA ladder as we have had problems with degradation.

• 40ul of 500ug/ml promega lambda HindIII digest (20ug in total)
• 280ul H2O
• 80ul loading dye

=400ul which was aliquoted into 4 tubes; 3 put in the freezer 1 in the fridge.

Metal Sensing Team

Introduction

Practical Outline

This is the initial list of tasks which need to be carried out by the end of the day:

1. Start the Bacillus subtilis transformation process ("Day of transformation" steps of protocol) in the morning.
2. Design primers for checking B. subtilis cotC integration
3. Plate out some TPA2 cells for production of competent Dh5-alpha E. coli cells
4. Run digests from yesterday (midi-prep digests) on gel
   1. If successful transform B. subtilis with midi-prep DNA
   2. If unsuccessful prepare another ligation attempt
5. Carry out a second attempt at digesting the 5 midi-prep samples from the 11/09/09 Lab Session, i.e. cotC-GFP-smtA, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3

Procedure

Starting Bacillus subtilis transformations

The team started the first stage of the Bacillus subtilis transformation process by carrying out the beginning steps of the "Day of transformation" section shown in the protocol. If the midi-prep test digest analysis shows that the ligations and the transformation of cotC-GFP-smtA in the pMUTIN4 plasmid have worked then some of the remaining midi-prep DNA can be used to transform B. subtilis when the time is right.

Designing primers for cotC BioBrick integration

It is key that if the team manage to transform Bacillus subtilis with the cotC-GFP-smtA BioBrick in pMUTIN4 plasmid, they can determine that the BioBrick has successfully integrated into the B. subtilis genome. We have designed the BioBrick so that it integrates within the cotC gene of the B. subtilis genome so any primers designed should flank this region. Unfortunately, due to time restrictions, we didn't manage to carry this step out; it will be carried out another day.

Plating out TPA2 cells

It has been noted that the stock of competent DH5-alpha E. coli cells stored in the -80C freezer has been greatly reduced. To generate some more competent E. coli cells, some TPA2 cells (DH5-alpha E. coli) from the stocks freezer were taken and spread on an LB + agar plate. This is to fulfil the first step of the "Pre-culture" stage in the "Preparing Competent Cells" protocol. The plate will then be grown overnight in the 37C incubator.

Analysing midi-prep digests

Analysing cotC, kinA, pGFP-rrnB, pMUTIN4 and pSB1AT3 midi-preps

July M T W T F S S     [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_July_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_July_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_July_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_July_2009&action=edit 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_July_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_July_2009&action=edit 6] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/7_July_2009&action=edit 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_July_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_July_2009&action=edit 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_July_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_July_2009&action=edit 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_July_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_July_2009&action=edit 13] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/14_July_2009&action=edit 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_July_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_July_2009&action=edit 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_July_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_July_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_July_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_July_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_July_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_July_2009&action=edit 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_July_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_July_2009 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_July_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_July_2009&action=edit 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_July_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_July_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_July_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_July_2009 30] [http://2009.igem.org/Team:Newcastle/Labwork/31_July_2009 31]

August M T W T F S S           [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/1_August_2009&action=edit 1] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/2_August_2009&action=edit 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_August_2009&action=edit 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_August_2009 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_August_2009 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_August_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_August_2009 7] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/8_August_2009&action=edit 8] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/9_August_2009&action=edit 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_August_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_August_2009 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_August_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_August_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_August_2009 14] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/15_August_2009&action=edit 15] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/16_August_2009&action=edit 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_August_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_August_2009 18] [http://2009.igem.org/Team:Newcastle/Labwork/19_August_2009 19] [http://2009.igem.org/Team:Newcastle/Labwork/20_August_2009 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_August_2009 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_August_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_August_2009&action=edit 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_August_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_August_2009 25] [http://2009.igem.org/Team:Newcastle/Labwork/26_August_2009 26] [http://2009.igem.org/Team:Newcastle/Labwork/27_August_2009 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_August_2009 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_August_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_August_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_August_2009&action=edit 31]

September M T W T F S S   [http://2009.igem.org/Team:Newcastle/Labwork/1_September_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_September_2009 2] [http://2009.igem.org/Team:Newcastle/Labwork/3_September_2009 3] [http://2009.igem.org/Team:Newcastle/Labwork/4_September_2009 4] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/5_September_2009&action=edit 5] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/6_September_2009&action=edit 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_September_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_September_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_September_2009 9] [http://2009.igem.org/Team:Newcastle/Labwork/10_September_2009 10] [http://2009.igem.org/Team:Newcastle/Labwork/11_September_2009 11] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/12_September_2009&action=edit 12] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/13_September_2009&action=edit 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_September_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_September_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_September_2009 16] [http://2009.igem.org/Team:Newcastle/Labwork/17_September_2009 17] [http://2009.igem.org/Team:Newcastle/Labwork/18_September_2009 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_September_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_September_2009&action=edit 20] [http://2009.igem.org/Team:Newcastle/Labwork/21_September_2009 21] [http://2009.igem.org/Team:Newcastle/Labwork/22_September_2009 22] [http://2009.igem.org/Team:Newcastle/Labwork/23_September_2009 23] [http://2009.igem.org/Team:Newcastle/Labwork/24_September_2009 24] [http://2009.igem.org/Team:Newcastle/Labwork/25_September_2009 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_September_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_September_2009&action=edit 27] [http://2009.igem.org/Team:Newcastle/Labwork/28_September_2009 28] [http://2009.igem.org/Team:Newcastle/Labwork/29_September_2009 29] [http://2009.igem.org/Team:Newcastle/Labwork/30_September_2009 30]