SBB11Ntbk-Xin Xin Lin
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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#
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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