Team:Duke/Notebook

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<p class="big"> Lab Notebook </p>
<p class="big"> Lab Notebook </p>
<p> From <a href="https://docs.google.com/document/d/1McU5sMTF2g2D6V6tyAHTmvbuNf3j_ZDRABgacDiQtTQ/edit#heading=h.w8ipel8cg1sr"> Google doc </a> </p>
<p> From <a href="https://docs.google.com/document/d/1McU5sMTF2g2D6V6tyAHTmvbuNf3j_ZDRABgacDiQtTQ/edit#heading=h.w8ipel8cg1sr"> Google doc </a> </p>
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Revision as of 21:18, 28 July 2014


Lab Notebook

From Google doc

April 2014
Month 1 of Project
sun mon tue wed thu fri sat
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30
May 2014
Month 2 of Project
sun mon tue wed thu fri sat
1 2 3
4 5 6 7 8 9 10
11 12 13 14 15 16 17
18 19 20 21 22 23 24
25 26 27 28 29 30 31
June 2014
Month 3 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6 7
8 9 10 11 12 13 14
15 16 17 18 19 20 21
22 23 24 25 26 27 28
29 30
July 2014
Month 4 of Project
sun mon tue wed thu fri sat
1 2 3 4 5
6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August 2014
Month 5 of Project
sun mon tue wed thu fri sat
1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September 2014
Month 6 of Project
sun mon tue wed thu fri sat
1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30
October 2014
Month 7 of Project
sun mon tue wed thu fri sat
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30 31

April Overview

Objectives

This is what we were trying to accomplish in April.

April 7

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw

PCR of Z4EV from pMN10

  • new oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p (diluted to 100uM)
  • 4 tubes with 0, 0.3, 0.6, 1 uL template
    • iGEM 3-step protocol with 65C anneal, 20 sec extend
  • Note: did not dilute template beforehand (do this in future)

Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)

  • Expected band size 715 bp
  • Control (no template) showed no band, lane 4 produced faint band
  • Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)

Next steps:

  • Run on gel to confirm (4/8)
  • digest PCR product and dCas9/Mxi1 with SpeI/NcoI to ligate (4/8)

April 8

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

Agarose Gel with Z4EV PCR products

  • PCR from 04/07/2014 of Z4EV from pMN10
  • Results (04/08/2014): Lanes 2 and 3 produced band at ~0.7-0.8 kb (agarose gel)
    • Expected band size 715 bp
    • Control (no template) showed no band, lane 4 produced faint band
    • Lanes 3 and 4 showed strong band at ~5-6 kb (template expected 5.1 kb)

PCR cleanup of Z4EV PCR products

  • PCR from 04/07/2014 of Z4EV from pMN10
  • Used only lanes 2 and 3 (successful from gel)
  • Concentration 32.4 ng/uL in 50 uL

Restriction Digest of Z4EV PCR product and TDH3-dCas9-Mxi1

  • Both with SpeI-HF and NcoI-HF in Cutsmart
  • PCR from 04/07/2014 of Z4EV from pMN10 -- 50 uL (entire product) in 65 uL reaction
  • dCas9 plasmid construct from CC 190ng/uL -- 20 uL in 65 uL reaction

PCR cleanup of Z4EV PCR digest product

  • 04/08 digest of 04/07 PCR
  • Results: Final concentration negligible, no DNA

Next steps:

  • Z4EV PCR again from pMN10
  • Digest of PCR in SpeI/NcoI
  • Gel extraction of digested TDH3-dCas9-MxiI

April 10

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

PCR of Z4EV from pMN10

  • Repeat of PCR from 4/7/14
  • Oligos SpeI-ZeEV5p and NcoI-Spc-Z4EV3p
  • Diluted pMN10 template to 1 ng/uL before use
  • 8 tubes: 0, 0.3, 0.5, 0.5, 0.7, 0.7, 1.0, 1.0 uL template in 50 uL reaction
    • iGEM 3-step protocol with 65C anneal, 20 sec extend
  • Results (see 4/11): expected bands present, but concentrations too low

Gel Extraction of backbone (-TDH3) from TDH3-dCas9-Mxi1 digest

  • Digest performed 4/8 with SpeI/NcoI
  • Expected band sizes: 10.5 kb (backbone, desired piece), 637 bp (TDH3, removed piece)
  • Obtained consistent band sizes, used 400 mg gel in extraction <
  • Gel picture:
  • Results: obtained 20 uL product at 55 ng/uL (froze for later use)

Next steps:

  • Run gel of PCR
  • Clean up PCR

April 11

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Matthew Faw

Agarose gel to analyze PCR of Z4EV from pMN10

  • PCR from 4/10
  • Expected band size: 715 bp
  • Showed band at expected size in all seven non-control lanes
  • Intensities appear low, increase in higher lanes (higher template conc.)
  • Gel picture:

PCR cleanup of Z4EV from 4/10 PCR

  • final concentration from 7 tubes: 25.8 ng/uL in 30 uL
  • Need to improve

Next Steps:

  • Redo PCR of Z4EV. Things to try:
    • Use previous cleanup as a template
    • use 1 uL enzyme instead of 0.5
    • slightly longer extension time
    • higher template concentration
  • PCR cleanup: Things to try:
    • Run PB buffer flow-through back through filter during step 7
    • let PE buffer sit in column (covered) while ethanol evaporates
    • let elution buffer sit on DNA for 2-3 minutes before spin

April 13 - Unfinished

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Garima Tomar

PCR of Z4EV from pMN10

  • Third attempt (previously 4/7, 4/10)
  • oligos SpeI-Z4EV5p and NcoI-spc-Z4EV3p
  • 8 identical tubes with 1.5 uL template (1 ng/uL stock) in 50 uL reaction
    • Used 1 uL polymerase (instead of 0.5 uL) per 50 uL reaction
    • iGEM 3-step protocol with 65C anneal, 1 min extension
  • Changes from previous: more template, more polymerase, longer extension

Results (Date):

  • ???

Next steps:

  • Run on gel
  • Clean up PCR with new notes (see 4/11), see if concentration is better

April 14

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

Agarose gel of Z4EV PCR

  • Z4EV from pMN10, 4/13 PCR
  • 2-log ladder and 8 identical reaction tubes
  • Expected band size ~715 bp
    • All lanes showed band 700-800 bp
    • band intensity much greater than previous attempts
  • Unable to take normal picture: used dark room and iPhone camera to photograph

PCR cleanup of Z4EV PCR from 4/13

  • Qiagen miniprep steps 7-10
  • 8 tubes into 1 final product
  • Modifications to protocol:
    • ran PB buffer through twice
    • let column sit, covered, for 10-15 min after PE buffer wash/discard step
    • let water for elution sit on column for 3 min before spin
  • Results: Obtained 30 uL product at 407.5 ng/uL (in gel fridge for immediate use)

Next Steps:

  • Restriction digest of Z4EV and PCR cleanup with SpeI and NcoI
  • ligation of Z4EV with dCas9 backbone (obtained 4/10)
  • transformation

April 15

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano

Restriction Digest of Z4EV-PCR with SpeI/NcoI

  • PCR from 4/13, cleaned 4/14
  • Cut with SpeI-HF and NcoI-HF in Cutsmart buffer
    • 30 uL PCR product (407.5 ng/uL) in 50 uL reaction
    • Incubated at 37C for 4.5 hrs

PCR Cleanup of Restriction Digest of Z4EV

  • Qiagen kit with modified methods described on 4/14
  • Results: Obtained 30 uL product at 248.1 ng/uL DNA (nanodrop)

Ligation of Z4EV PCR and dCas9-MxiI backbone

  • Z4EV from pMN10, digested with NcoI and SpeI (4/15)
  • dCas9-MxiI backbone digested with NcoI and SpeI, extracted 4/10
  • 3:1 molar ratio backbone:insert
    • 20 uL total reaction - 15.3 uL backbone (55 ng/uL), 0.7 uL insert (248.1 ng/uL)
  • Left in cold room overnight in 16C heat block

Next steps:

  • Transform ligated plasmid into E. coli, clone, then miniprep.

April 16

Objective: Create Z4EV-dCas9-Mxi1 construct
Matthew Farnitano and Charlie Cooper

Transformation of ligated Z4EV-dCas9-MxiI plasmid into E. coli

  • Ligation left overnight for 22 hrs
  • Heat shock protocol with chemically competent E. coli
    • 1 sample plus 1 no-insert control
    • grown in SOC medium, plated on LB+Amp plates
  • Results (Charlie Cooper) (4/17): No colonies on either plate
    • Possibly used ineffective cells

Next steps:

  • Digest and gel extraction of backbone
  • Ligation and Transformation

May, June Overview

Objectives

This is what we were trying to accomplish in May and June.

May 29

Objective: Prepare Chemically competent E. Coli
Matthew Faw

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

  • Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

  • Transformation

June 1

Objective: Transform Chemically competent E. Coli (CCEC)
Matthew Faw

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

  • Followed Charlie’s Cloning Protocols
    • Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
  • Plated the transformed DNA and put in incubator at 37C
  • Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

  • Look at plates, compare cultures

June 2

Objective: Redo yesterday’s transformation
Matthew Faw

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

  • Followed Charlie’s Cloning Protocols
    • Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
  • Plated the transformed DNA on 6 separate plates, put in 37C overnight
  • Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

  • Look at plates, compare cultures, etc.