SBB11Ntbk-Xin Xin Lin

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Xin Xin Lin 15:28, 21 April 2011 (EDT)

4/26/2011 & 4/28/2011:

Worked on data analysis, interpretation of results, & worked on slide presentation

See Rishi's Notebook for Graphs



12PM, Rishi gets data from Tecan & formats data for initial graphs

Tecan Output Media:original_data.csv

Formatted data Media:formattedData.csv



4/11/2011- 5:30PM, Jessica started 3mL cultures of ToxR, Violacein, Control

10AM, Pipetted 2mL LB+spec in 39 wells of block

     Added 200uL cells to each well, 13 wells correspond to 1 type(either ToxR, v or control)

11:15AM, Placed blocks in 37C shaker in Anderson Lab

1:00, Anand & I added arabinose & 100uL cells to block

     Ran Tecan for 20hours measuring OD every 6 minutes



Rishi, Jessica, & I came in at 8AM to run Tecan experiment- Waited 1.5 hours, but no one was in lab.



~5PM- Jessica started O/N cultures for 3 samples- In 37C shaker in Anderson Lab _________


12pm: Rishi moved cells from incubator to 4C fridge in Anderson Lab, next to incubator

All plates had 100s of colonies--> transformation worked



Transform (heat shock) pBCA1766-BCA1144 into MC1061- Control w/ RFP

                      pBca1256-bdc004 (named bdc022)- ToxR
                      pBca9523-jtk2914- VioABCDE

Follow E. coli Transformation Protocol

1. Thaw a 200 uL aliquot of competent cells on ice

2. Add 50 uL of water to the cells (if greater volume is desired)

3. Add 30 uL of KCM to the cells- did not add KCM, volume cells too small

4. Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)- did not dilute miniprep product

5. Add 70 uL of the cell cocktail to the ligation, stir to mix- added 2uL miniprep plasmid/10uL diluted & 50uL cells

6. Let sit on ice for 10 min- waited 7 min.

7. Heat shock for 90 seconds at 42 (longer incubation may work better)

8. Put back on ice for 1 min

9. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour- used LB media

10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight- plate 50uL on Spec plates


3/15/2011 & 3/17/2011:

Group Project: Evaluate Toxicity of ToxR & Violacein

Design Protocol for Project- See Google Doc & Group Notebook (Team ToxRVio)



Sequencing Analysis of Constructed Plasmids

pBca1766 jtk2791

pBca9523 jtk2914

pBca1766 jtk2979

pBjh1601KC sbb1117

pBjh1601KC sbb1129

pBjh1601KC sbb1105

pBjh1601KC sbb1110

pBjh1601KC sbb1139

AB1 pBjh1601KC-sbb1117- Perfect

       BB1- Perfect

AF2 pBjh1601KC-sbb1129- Perfect

BB2 pBjh1601KC-sbb1139- Perfect

       AB2- Perfect

BE2 pBjh1601KC-sbb1110- Perfect

AE2- Perfect

AB3 pBjh1601KC-sbb1105- Perfect

BB3- Perfect

AC5 pBca1766-jtk2979- Partial Perfect

AD5 pBca1766-jtk2791- Partial Perfect

AE5 pBca9523-jtk2914- Bad Read, Only see very first part of sequence

Xin Xin Lin 13:29, 10 March 2011 (EST)

1. Ran Gel 3/8/11 after Soaking in TAE Buffer

  Verified Bands in all replicates of P_fadR
  Bands in replicates of P_cspG very faint

2. Selected 2 Clones of Each Part for Sequencing

  Used Replicate 1&2 of P_fadR & P_cspG

3. P_cspE Needs to be Redone

  E. coli did not transform with plasmid (pink colonies or no growth)

Xin Xin Lin 14:05, 8 March 2011 (EST)

1. Analytical Digests (Mapping)

  8 Miniprep Products- 4 Replicates P_fadR & 4 Replicates P_cspG
  Mapping Master Mix:   x10
  4uL ddH2O             ->   40uL ddH2O
  1uL 10x NEB Buffer 2  ->   10uL 10x NEB Buffer 2
  0.5uL EcoRI           ->   5uL EcoRI
  0.5uL BamHI           ->   5uL BamHI
  Add 6uL Master Mix to 4uL Miniprep Plasmid
  Incubate 30min. @ 37 degrees C

2. Run Analytical Gel

  Gel #7- Lanes 6-13
  Image Gel & Calculate Fragment Sizes

3. Submit for Sequencing

Xin Xin Lin 15:01, 3 March 2011 (EST)

1. Miniprep Purification of DNA

  4 Transformed E. coli Colonies Picked/Plate & Cultured
  -P_cspE Plate had very few colonies & several pink/red colonies (Not Transformed)
  -P_cspE 1 & 2 Cultures Cloudy Pink, P_cspE 3 & 4 Cultures Clear (No Growth)
  -Miniprepped only P_fadR 1-4 & P_cspG 1-4
  Pellet @ Max Speed for 30sec & Dump Supernatant
  Add 250mL Suspension Buffer P1- Resuspend (Vortex)
  Add 250mL Lysis Buffer P2- Pipet Up & Down
  Add 350mL Precipitation (Acid) Buffer N3- Shake Up & Down
  Spin 5 min. & Pour supernatant into Miniprep Columns (Blue)
  Spin 15 sec & Remove Liquid
  Add 500mL Wash Buffer PB
  Spin 15 sec & Remove Liquid
  Add 750mL Wash Buffer PE- Remove salts from resin
  Spin 15 sec & Remove Liquid
  Spin 90 sec to dry
  Elute w/ 50uL ddH2O in Clean 1.5mL Eppendorf Tube

Xin Xin Lin 14:51, 1 March 2011 (EST)

1. Ligation

  Add 6.5uL ddH2O, 1uL T4 Ligase Buffer, 1uL pBjh1601KC Vector, 1uL Insert (Digested PCR Parts), & 0.5uL T4 DNA Ligase
  Incubate for 30 minutes @ Room Temp. in 500mL Eppendorf Tubes

2. Prepare Lefty E. coli Competent Cells- Yellow Test Tubes

  Add 50uL ddH2O & 30uL KCM to 200uL Cells after thawing
  Leave on ice

3. Transformation by Heat Shock

  Add 70uL cell cocktail to ligation reactions & stir
  Heat shock for 90sec @ 42 degrees C & Ice for 1 min.
  Recover w/ 100uL LB Media (Flame bottle & tips) & shake for 1 hour @ 37 degrees C
  Plate 70+uL mixture on Kan Plate w/ Glass Beads
     Green Stripes=Kan (Kanamycin)- KC vector has KnR & CmR, can use either plate
     Blue Stripes=Cam (Chloramphenicol)
  Incubate @ 37 degrees C overnight

Xin Xin Lin 13:44, 24 February 2011 (EST)

1. Zymo Gel Purification Cleanup

  Transfer all 600uL Dissolved Gel in ADB Buffer to Zymo Column
  Spin for 15 sec @ full speed & discard waste
  Add 200uL A4 Wash Buffer, Spin, & Discard x2
  Spin for 90 sec @ full speed to dry
  Elute in 8uL ddH2O (Amount of Digested PCR Product Used)

Xin Xin Lin 13:45, 22 February 2011 (EST)

1. EcoRI/BamHI Digestion

  8uL Eluted PCR Product in 1uL NEB 2 Digestion Buffer w/ 1uL EcoRI & 1uL BamHI
  Incubate in 37 degrees C Thermocycler for 1 Hour

2. Run Preparative Gel

  Cut out Digested Bands to Gel Purify (Lanes 2-4 of Gel B)
  Place gel in 600uL ADB Buffer & Melt at 55 degrees C

Xin Xin Lin 15:01, 18 February 2011 (EST)

Work done on 2/17/11:

1. Run Analytical Gel of PCR Product

  2uL PCR Product in 5uL Loading Buffer
  Load 5uL DNA Ladder & 8uL PCR Samples- Run at 180V

2. Upload Gel Image- fadR, cspE, & cspG in lanes 4,5,&6 of Analytical Gel Image 1

  PCR Products Visible- ~767 & 2 432bp Bands

3. Regular Zymo Cleanup- Followed Protcol

  Eluted Cleaned DNA in 33uL ddH2O- Stored in 140L Box A

Xin Xin Lin 14:07, 15 February 2011 (EST)

1. PCR with P_fadR, P_cspE, & P_cspG

  Diluted fadR-F & fadR-R Oligos with 243 and 298uL ddH2O to 100uM
  Made 1:10 Dilution- 1uL 100uM Oligos in 9uL ddH2O to 10uM Dilution
  P_cspE & P_cspG Oligos already diluted to 10uM- ss39f&r for P_cspE and ss40f&r for P_cspG
  Used E.coli MG1655 Genomic DNA as Template- Used Expand Buffer & Expand Polymerase 1
  PCR Products should be 767, 432, & 432bp respectively- Used 2K55 PCR Program b/c PCR Products<2000bp