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Formal Lab Session - 22nd September 2009

Overview

• Metal Sensor Team -

• Stochastic Switch Team -

• Sporulation Tuning/Chassis Team -

Metal Sensor Team

We carried out the following tasks:

• Digested cotC-GFP-smtA PCR products with BamHI and HindIII (FastDigest) and allowed them to incubate for 1 hour under 37C.

• Conducted PCR clean-up using GenElutes PCR clean-up kit

• Digested cotC-GFP-smtA with BamHI and HindIII

• Carried out FastLigations with the following concentrations:

1. 2ul of ligase buffer
2. 2.5ul of vector DNA (i.e. pMUTIN4)
3. 14.5ul of insert DNA (i.e. cotC-GFP-smtA)
4. 1ul of ligase

Please note there were three reactions: 1) vector + insert + ligase, 2) vector + ligase and 3) vector + NO ligase.

• We also started to make some competent E. coli cells by carrying out the 'Culture' step in Phil Aldridge's preparing competent cells protocol

Stochastic Switch Team

Today we transformed E.coli JM109 cels with the overnight ligation product from yesterday using the promega protocol. We used the following controls:

• Tube 1: Backbone + insert + ligase (proper ligation)
• Tube 2: Backbone + ligase (control for background ligation level)
• Tube 3: Backbone + insert (control for ligase effectiveness)

These were left in the incubator for an hour and then plated out (2 plates per tube 200ul and 50ul) onto Amp + Tet LB plated and put in the 37C incubator overnight.

Today again we redid the digests of the sac miniprep however again there was no DNA present.

Sporulation Tuning/Chassis Team

Introduction

• After overnight culture of cells for Midi Prep, today we need Midi Prep L1 and L2 plasmid and digest the plasmid to check the Midi prep result.

Experiment procedure

Midi prep L1(pSB1AT3:cwlJ) and L2(pSB1AT3:cwlJ:sleB)

• Before we carried out Midi Prep, we take out 1ml of cell mix and put them into 1.5ml eppendorf tube to keep in fridge.
• 50ml of cells were used for Midi Prep.
• Midi prep processed by following the standard protocal of Plasmid Midi Prep Kit(Sigma).

Digest Midi prep result

• Using the same reaction volume as day 18/09/09

Conclusion

• Both Midi prep for L1 and L1 are right plasmid.
• Nano dorp of our fragments and plasmid.

 cwlJ:sleB fragment 53.5ng/ul
 (after PCR using primer cwlJsleBForward and cwlJsleBReverse)
   
 pMutin4 after digestion with HindIII and BamHI 24.9ng/ul

 L1 Midi (plasmid pSB1AT3:cwlJ)  76.9ng/ul

 L2 Midi (plasmid pSB1AT3:cwlJ:sleB) 55.0ng/ul

 kinA fragment after digest(EcoRI and NheI) and gel extraction  6.1ng/ul

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]

October M T W T F S S       [http://2009.igem.org/Team:Newcastle/Labwork/1_October_2009 1] [http://2009.igem.org/Team:Newcastle/Labwork/2_October_2009 2] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/3_October_2009&action=edit 3] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/4_October_2009&action=edit 4] [http://2009.igem.org/Team:Newcastle/Labwork/5_October_2009 5] [http://2009.igem.org/Team:Newcastle/Labwork/6_October_2009 6] [http://2009.igem.org/Team:Newcastle/Labwork/7_October_2009 7] [http://2009.igem.org/Team:Newcastle/Labwork/8_October_2009 8] [http://2009.igem.org/Team:Newcastle/Labwork/9_October_2009 9] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/10_October_2009&action=edit 10] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/11_October_2009&action=edit 11] [http://2009.igem.org/Team:Newcastle/Labwork/12_October_2009 12] [http://2009.igem.org/Team:Newcastle/Labwork/13_October_2009 13] [http://2009.igem.org/Team:Newcastle/Labwork/14_October_2009 14] [http://2009.igem.org/Team:Newcastle/Labwork/15_October_2009 15] [http://2009.igem.org/Team:Newcastle/Labwork/16_October_2009 16] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/17_October_2009&action=edit 17] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/18_October_2009&action=edit 18] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/19_October_2009&action=edit 19] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/20_October_2009&action=edit 20] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/21_October_2009&action=edit 21] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/22_October_2009&action=edit 22] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/23_October_2009&action=edit 23] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/24_October_2009&action=edit 24] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/25_October_2009&action=edit 25] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/26_October_2009&action=edit 26] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/27_October_2009&action=edit 27] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/28_October_2009&action=edit 28] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/29_October_2009&action=edit 29] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/30_October_2009&action=edit 30] [http://2009.igem.org/wiki/index.php?title=Team:Newcastle/Labwork/31_October_2009&action=edit 31]