SBB11Ntbk-Xin Xin Lin

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

4/4/2011 & 4/6/2011:

Group Project: Evaluate Toxicity of ToxR & Violacein

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

https://docs.google.com/document/d/1G527lFDDkvtGUGSL--SO19OVThIQupm5D3xtnC9hFLI/edit?hl=en&pli=1#

_________

3/12/2011 & 3/14/2011:

Sequencing Analysis of 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