SBB11Ntbk-Xin Xin Lin: Difference between revisions
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==[[User:Xin Xin Lin|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 | |||
_________ | |||
4/13/2011: | |||
12PM, Rishi gets data from Tecan & formats data for initial graphs | |||
Tecan Output [[Media:original_data.csv]] | |||
Formatted data [[Media:formattedData.csv]] | |||
_________ | |||
4/12/2011: | |||
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 | |||
_________ | |||
4/8/2011: | |||
Rishi, Jessica, & I came in at 8AM to run Tecan experiment- Waited 1.5 hours, but no one was in lab. | |||
_________ | |||
4/7/2011: | |||
~5PM- Jessica started O/N cultures for 3 samples- In 37C shaker in Anderson Lab | |||
_________ | |||
4/6/2011: | |||
12pm: Rishi moved cells from incubator to 4C fridge in Anderson Lab, next to incubator | |||
All plates had 100s of colonies--> transformation worked | |||
_________ | |||
4/5/2011: | |||
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) | |||
https://docs.google.com/document/d/1G527lFDDkvtGUGSL--SO19OVThIQupm5D3xtnC9hFLI/edit?hl=en&pli=1# | |||
_________ | |||
3/10/2011: | |||
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 | |||
==[[User:Xin Xin Lin|Xin Xin Lin]] 13:29, 10 March 2011 (EST)== | ==[[User:Xin Xin Lin|Xin Xin Lin]] 13:29, 10 March 2011 (EST)== |
Latest revision as of 14:31, 2 May 2011
~~!~~
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
_________
4/13/2011:
12PM, Rishi gets data from Tecan & formats data for initial graphs
Tecan Output Media:original_data.csv
Formatted data Media:formattedData.csv
_________
4/12/2011:
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
_________
4/8/2011:
Rishi, Jessica, & I came in at 8AM to run Tecan experiment- Waited 1.5 hours, but no one was in lab.
_________
4/7/2011:
~5PM- Jessica started O/N cultures for 3 samples- In 37C shaker in Anderson Lab _________
4/6/2011:
12pm: Rishi moved cells from incubator to 4C fridge in Anderson Lab, next to incubator
All plates had 100s of colonies--> transformation worked
_________
4/5/2011:
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)
https://docs.google.com/document/d/1G527lFDDkvtGUGSL--SO19OVThIQupm5D3xtnC9hFLI/edit?hl=en&pli=1#
_________
3/10/2011:
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