SBB10AssayTeam1-LabNotebook

4/28/2010

Media:elisa-data.xls

Absorbance vs. concentration of T4 Ligase

Absorbance vs. T4 Ligase with various tags

Suggestions for ELISA

1. Problem: Absorbances remain unchanged/increases with decreasing protein concentration

Possible cause: Protein concentrates saturates available antibody
Solution 1: Reduce protein concentrations below 1/10000X
Solution 2: Perform a second purification step
Solution 3: Increase antibody concentrations available to bind protein (primary detecting (anti-His) or primary capture (anti-Myc))

2. Problem: Absorbances remains unchanged/increases with decreasing protein concentration

Possible cause: Protein concentrates saturates available antibody.
Solution 1: Reduce protein concentrations below 1/10000X
Solution 2: Perform a second purification step.
Solution 3: Increase antibody concentrations available to bind protein (primary detecting (anti-His) or primary capture (anti-Myc))

4/26/2010

Did ELISA. Left lab around 5 pm; last step performed was incubating 1h after adding varying concentrations of T4 ligase to 24 wells.

Protocol (roughly):

1. Dilute anti-Myc antibody (1000X) to 1X in TBST, plate 100 uL into well
2. Incubate at RT for 1h
3. Do three plate washes (TBST with 0.1% Tween)
4. Put blocking buffer (2% BSA, in TBST) 100 uL in wells, incubate 1h at RT
5. Do three plate washes
6. Put 100 uL T4 Ligase at various concentrations (diluted in blocking buffer) (see attached spreadsheet) into wells
7. Incubate 1h at 37C
8. Do three washes
9. Put anti-His antibody conjugated to HRP (10000X) 100uL, dilute to 1X in blocking buffer, 1h at 37C
10. Do three washes
11. Put detection 3,3’,5,5’-Tetramethylbenzidine (TMB) solution 100uL
12. Incubate at 37C for 15 min
13. Detect with spectrometer at 450 nm

4/22/2010

1. Get column, cut tip, tape it to stabilize somewhere high, place waste column beneath the column.
2. Pour in lysate + beads
3. Collect drips
4. Wash with ice cold PBS (20 mL) (N x 0.5 mL) eject PBS against the column wall to wash lysate off the wall.
5. Elute with 4-5 times 1 mL volumes of 300 mM imidazole in PBS
6. Put eluant into dialysis column, add ice cold PBS to column, then shake in a speed of 5000mph with 4°C for 30 min, then change to fresh PBS, repeat shaking and change media for several times.
7. add ligase, purify and run in gel (Mike did this).

4/21/2010

Notes for 4/21/2010

• General protocol for protein purification:

1. Pick starter colony
2. Grow starter culture
3. Put in flask
4. Grow to midlog
5. Induce arabinose
6. Grow to saturation, have protein
7. Transfer to centrifuge tube (100 mL) x 2 - stuff here on is on ice (4C)
8. Spin 5 min at 6000 rpm
9. Dump supernatant, leave pellet
10. 10 mL of resuspension buffer (TBS), resuspend and combine in 50 mL conical flask
11. Sonicate (probe) - 1 minute (is it a program?). DO pulses.
12. Spin to remove debris
13. Transfer supernatant to fresh tube (pour). Will be translucent.
14. Add 400 uL of Ni resin
15. Agitate at 4C 1h to overnight

• Tomorrow

1. Get column
2. Pour in lysate + beads
3. Collect drips
4. Wash with PBS (20 mL) (4 x 5 mL)
5. Elute with 4-5 1 mL volumes of 300 mM imidazole in PBS
6. Put eluant into dialysis bag
7. Let dialyze overnight on ice (4°C)

4/19/2010

• analyze TECAN data, maximize TECAN protocol
  • TECAN data showed significant edge effects. Recommended to not use outer rows and columns
  • I have no clue how to derive the actual growth rate. Tried some logistic equations (a*b)/(a-(b-a)*Exp(-c*t)) and nonlinear regression but my system keeps blowing up, regression looks bad, etc.
  • There are a lot of forms of logistic equations you can use. E.x. K / (1 + exp(a + b*x), the differential form dN/dt = r*N(K-N)/K, etc. If anyone has a copy of Mathematica or SPSS, try to play around with it.
• develop ELISA protocol

4/15/2010

• materials: 20% 100X arabinose, LB media and 96 well plate for TECAN
• dilute arabinose to 1X, 50ul LB in each well. 1ul of saturated cell mixture.
• Plate: http://spreadsheets.google.com/ccc?key=0AvbXytCFRyFCdGJNVXhzNTUtWTgtZFlkYmZGTVQxT2c&hl=en
• by accident, added two uls of different cells to F3

TECAN

A TECAN Safire II machine at Architecture et Fonction des Macromolécules Biologiques

Running a TECAN analysis:
The procedure below only works for black 96-well flat bottom plates.

1. Turn TECAN power on (wait for light to stop flashing)
2. Open XFluor4 Safire II XLS spreadsheet
3. Load plate:
   1. Have 96-well plate with media+cells
   2. Goto XFluorSafireII menu > movements > out
   3. Load plate
   4. Goto XFluorSafireII menu > movements > in
4. Load the program to run:
   1. Select Multi Labeling Kinetic
   2. Load multi labeling kinetic parameter > "\iGEM 2007\My Documents\Weston\gabe's experimental folder\kinteticmodified_no gfp reading.mps"
   3. Click "Run"
5. Wait for data collection. Operation can only be cancelled when machine is performing measurements.

4/14/2010

Picked 3 colonies from the 3 different constructs. Transformation efficiency is, of course, insane because there was no ligation performed.

jtk2559 (2599?)

jtk2578

jtk2619

Ugh, image markup is confusing. Anyways, refer to the edit page for code to do the above.

4/12/2010

• Trial run for transformation. Obtained vectors.

1. jtk2559-jtk2164 K
2. jtk2619-jtk2164 K
3. jtk2578-jtk2164 K

• Transformation protocol:

1. Add:
   1. plasmid 0.5 uL
   2. cells (MC1061 pir+) 10 uL
2. Add to cells:
   1. KCM 1.5 uL
   2. Water 2.5 uL
3. Use normal heat shock
   1. Did @ 42C for 120 sec
4. Rescue (rescued 40 min)
5. Plate