Team:Toronto/Notebook

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Notebook

'' The prefect lab notebook is a noble goal that very few achieve. But we must all do our best.''

JUNE

Week one

15/06/15

• Prepared 8 x 500mL bottles of LB media
• 34 x 1mL Chloramphenicol (CAM) stocks
• 500mL of SOC media
• Made LB plates
• Made 70% EtOH

16/06/15

• Made SOC media
• Obtained DH10 β cells

17/06/15

• Bacterial transformation of RFP to test CAM stocks
• Mark made more LB and Agar
• Made overnight cultures of DH10 β

18/06/15

• Prepared GYT for making electrocompetent cells
• Prepared LB and Agar
• Observed growth of plated RFP
• Made overnight cultures of RFP from plates

19/06/15

• Prepared 40% glycerol for glycerol stocks
• Made glycerol stocks of MG1655 and RFP with pSB1C3 backbone
• Made chemically competent cells.

Week Two

22/06/15

• Prepared TSS Buffer for making chemically competent cells
• Made King's B media plates
• Prepared electrocompetent DH10 β
• Overnight cultures of transformed DH10β
• Made LB plates with CAM

23/06/15

• Miniprep of cultured bacteria
• GLycerol stocks of RFP plasmid
• Casting of the gel for Gel Electrophoresis, as well as loading dye preparation
• Made chemically competent cells, stored at -80C

24/06/15

• Made SOC media
• Bacterial transformation
• Preparation for gel electrophoresis.

25/06/15

• Prepared two overnight cultures of Pseudomonas putida F1 (PpF1) in King's B media
• Plated PpF1 onto LB and 2 X King's B plates
• Obtained:
  • pDTG601 - with todC1C2BA insert
  • pDTG602 - with todC1C2BAD insert
  • pDTG603 - with todC1C2BADE insert
• Stored obtained bacteria in the freezer

26/06/15

• Made 70% glycerol stocks of PpF1
• Bacterial transformation with RFP

Week Three

29/06/15

• Made LB + Agar
• Plated DH10 β

30/06/15

• Transportation of Dangerous Goods (TDG) training

01/07/15

• Canada Day - Holiday

02/07/15

• Restriction enzyme digest for Interlab study
• Prepared LB + Amp plates

03/07/15

• Gel Electrophoresis

JULY

Week Four

06/07/15

07/07/15

• gel electrophoresis of Device1 A and Device1
• ligation of device 1

08/07/15

• gel electrophoresis of interlab device 2

09/07/15

• Bacterial transformation with interlab device 2
• Plated transformed bacteria
• Prepared Ampicillin stocks
• Prepared 500mL of LB + Ampicillin (AMP) and stored at -4C
• Prepared stocks of 70% Glycerol

10/07/15

• Miniprep of RFP and pDTG601
• Gel electrophoresis of pDTG601 and RFP

Week Five

13/07/15

• Resuspended todF insert forward in 311uL of nuclease free water to a final 100uM solution
• Resuspended todE insert forward in 311uL of nuclease free water to a final 100uM solution
• Resuspended todF insert reverse in 300uL of nuclease free water to a final 100uM solution
• Resuspended todF insert forward in 297uL of nuclease free water to a final 100uM solution
• Resuspended todE insert in 100uL of nuclease free water

• Resuspended todF insert in 100uL of nuclease free water

• PCR amplified todE insert and todF insert using FroggaBio 2X Taq FroggaMix

• Ran PCR purification of TodE and TodF according to the PureLink PCR Purification protocol.
• Gel Electrophoresis of TodE and TodF PCR products.

14/07/15

• Prepared 250mL of SOC media
• Prepared 25 LB + CAM plates
• Gel electrophoresis of TodE and TodF PCR products

15/07/15

• PCR amplified todE insert using FroggaBio 2X Taq FroggaMix
• PCR purification of TodE
• ran gel electrophoresis of TodE
• Matt got stuck in the freight elevator.

16/07/15

17/07/15

• Gradient PCR for todE using FroggaBio 2X Taq FroggaMix

Week Six

20/07/15

• Gel electrophoresis
• Cast SDS-PAGE gels

21/07/15

• Ran SDS-PAGE on RFP lysate
• Made 40% glycerol stocks

• Made TSS Buffer

  22/07/15

• Made chemically competent E.coli DH10β

23/07/15

• Performed bacterial transformation to test old batch of chemically competent cells against the newly made ones
• A - old chemically competent cells with RFP + AMP resistence
• B - new chemically competent cells with RFP + AMP resistence
• Control - new chemically competent cells with RFP + CAM resistence
• Ran day 2 of the cell lysis protocol of RFP and pDTG601 and pDTG603

24/07/15

• Performed Synbiota protocol for RDP assembly of GFP and aCP.

Week Seven

27/07/15

• Prepared ligase for the P&P summer camp
• Prepared lysozyme stocks
• Performed day 3 of the cell lysis protocol
• Prepared day 1 of the cell lysis protocol

28/07/15

• Preparation for SDS-PAGE, and gel Casting
• Performed day 2 of the cell lysis Protocol

29/07/15

• Performed day 3 of the cell lysis Protocol
• PCR amplification of the pSBC13 backbone
• PCR purification of amplified backbone
• Gel electrophoresis of the pSBC13 backbone

30/07/15

• Performed the PCR Protocol for creation of RDP parts with TodE and TodF inserts, using NEB Phusion polymerase.

31/07/15

• Performed PCR purification the Synbiota PCR TodE and TodF
• Gel electrophoresis of PCR purified Synbiota TodE and TodF

AUGUST

Week Eight

03/08/15

• PCR amplification of pSB1C3 TodF using NEB Phusion polymerase

04/08/15

• PCR purification of TodF and pSBC13
• Synbiota BsaI digestion of TodE
• Gel electrophoresis of BsaI digested TodE, pSB1C3 backbone, todF

05/08/15

• Synbiota PCR amplification of TodF and PCR of pSB1C3 backbone using Phusion polymerase
• PCR purification
• Miniprep of RFP

TodF Gel - Synbiota August 5 TodE Gel - Synbiota August 5 Gel Backbone - Synbiota August 5 #### 06/08/15 Gel electrophoresis of TodF, with IDT todF as a control TodF Gel Digested - Synbiota August 6 TodF Gel - Synbiota August 6 #### 07/08/15 Gel electrophoresis of the pSB1C3 backbone Synbiota RDP assembly of TodE and aCP Re-tested competent cells with RFP ### Week Nine #### 10/08/15 Ran SDS-PAGE with RFP, pDTG601, pDTG603 lysates Observed results - high RFP expression Preparation for Ligase Chain reaction (LCR) #### 11/08/15 Miniprep of RFP PCR amplification of pSB1C3 backbone with NEB Phusion polymerase. PCR purification of the backbone Made chemically competent cells #### 12/08/15 Gradient PCR amplification of the pSB1C3 backbone using NEB Phusion polymerase and NEB Q5 High Fidelity polymerase (with and without GC enhancer). Performed DPN1 Digest and PCR purifications of amplified pSB1C3 Protocol #### 13/08/15 Performed bacterial transformations with miniprepped RFP and resuspended RFP (with both old and new competent cells, and old and new TSS buffer). All grew, miniprepped RFP and new TSS work best. Gel electrophoresis of PCR pSB1C3 backbone with Q5 polymerase, and with Q5 polymerase and GC enhancer. Performed LCR with TodE and TodF inserts Performed PCR of TodF with Phusion polymerase. Performed bacterial transformations with purified PCR products. #### 14/08/15 Made 500mL of LB + Agar made 70% EtOH Made 1X TAE PCR purification of amplified Synbiota TodF Gel Electrophoresis of Synbiota TodF ### Week Ten #### 17/08/15 PCR of the pSBC13 backbone with new primers, using NEB Q5 polymerase Gel electrophoresis of LCR TodE and TodF products DPN1 digest and PCR purification of the pSB1C3 backbone Gel electrophoresis of the pSB1C3 backbone #### 18/08/15 Performed and LCR reaction with TodE and TodF inserts Performed Synbiota assembly of TodE and aCP. Performed bacterial transformation with LCR and Synbiota products, with RFP as a control. #### 19/08/15 #### 20/08/15 Synbiota assembly of GFP and aCP Bacterial transformation with GFP, aCP and RFP as a control. #### 21/08/15 ### Week Eleven #### 24/08/15 Gel electrophoresis of Synbiota aCP and GFP #### 25/08/15 Performed an LCR reaction with TodE and TodF inserts. Gibson assembly of TodE as well as with TodE and TodF insterts #### 26/08/15 Gel electrophoresis of Gibson products of TodE and TodE & TodF #### 27/08/15 #### 28/08/15 Gel electrophoresis of Gibson products of TodE and TodE & TodF ## SEPTEMBER ### Week Twelve #### 31/08/15 Bacterial transformation with LCR products, RFP as a control PCR of Synbiota TodE with Phusion polymerase Synbiota assembly of aCP and TodE #### 01/09/15 Made 1xTAE Bacterial transformation with LCR remnants, aCP, todE, RFP. Gel electrophoresis of Synbiota TodE and aCP Overnight cultures of LCR products, TodE, RFP #### 02/09/15 Gibson assembly of TodE and TodF inserts. 1mL SOC aliquot stocks Transformation with aCP, TodE, RFP #### 03/09/15 Miniprep of LCR products, todE, RFP Synbiota assembly of todE and aCP #### 04/09/15 Synbiota assembly of TodE and aCP Bacterial transformation with TodE from Synbiota and with TodE from Gibson assembly, Gibson assembled TodE and TodF ### Week Thirteen #### 07/09/15 Labour Day #### 08/09/15 gel electrophoresis for TodF of Gibson assembly TodF Gel - Gibson September 8th #### 09/09/15 Gel electrophoresis of TodF for Gibson assembly Miniprep of RFP, TodE and aCP Bacterial transformations with Gibson TodEF plasmids, Gibson todE plasmid, RFP and LCP products TodF Gel - Gibson TodE - September 9 #### 10/09/15 Bacterial transformation with diluted Gibson products #### 11/09/15 Synbiota assembly of TodE Transformation with LCR product ### Week Fourteen #### 14/09/15 LCR of TodE and TodF

15/09/15

• Gel electrophoresis of LCR products

16/09/15

• Gibson assembly of TodE and TodF and of TodE
• Bacterial transformation with LCR products and Giboson products

17/09/15

• Observed Growth of Gibson assembled TodE and prepared overnight cultures.

18/09/15

• Miniprep of Gibson assembled todE plasmid
• Preparing part for shipping to iGEM HQ, used a concentrator to speed vacuum our part.
• Performed two restriction enzyme digests to verify for the presence of TodE
• First digest with XhoI and XcmI produced appropriate band lengths.
• The Second Digest was with RsaI and PvuII also confirmed TodE presence.