Worked until 8PM

did gel in the morning, and carried out experiment... different time points, sonification, luciferase assay

ahh so much stuff  ¶ 11:02 PM

  Wednesday (yesterday)

after plasmid maxi prep tried to find the concentration of DNA with a spectrometer... low readings... going to do a gel tomorrow   ¶ 8:37 AM

  11AM began transfection with 1ul lipofectamine and 1ug 3TP per well

Total

108 wells ~115 wells
115ul lipfectamine in 11.5ml media in 15ml tube
164ul 3TP in 17.25ml media in 50ml tube

repeating pipette 250ul per well

1PM Qiagen Plasmid Maxi Prep for HLREV AB and HLREV   ¶ 1:21 PM

  Tuesday, Yesterday

Cultured bacteria for 2 genes, wild and mutant

2ml LB in test tubes, w/ 2ul ampicillin

200ul of that into 250ml w/ 250ul ampicillin

for each gene  ¶ 8:24 AM

  cell concentration too low to continue transfection... waiting until tomorrow!!!

arggg  ¶ 9:53 AM

  black speckles inhibiting cell grow

split the bes21 cells and plated them again

resuspended in 5ml

0.5ml in new T75

2.5ml in 60ml media for 120 wells, 500ul cell solution per well

500ul media + 500ul cel solution = 1ml solution/well
  ¶ 1:27 PM

  air flow alarm is going crazy, and its 10000 degrees in the lab... and the alcohol is shooting out of the squirt bottle by it self.... ahhh   ¶ 10:05 AM

  went to the lab this afternoon, sunday, 2pm, note said not to continue with experiment  ¶ 12:12 AM

  went in today, got there at 9... checked cells, density was low, changed the media, went home, going to do the experiment tomorrow, sunday, at 2pm  ¶ 3:53 PM

  Cells are not ready for egcg treatment today, too few cells, have to wait until tomorrow (saturday).   ¶ 10:25 AM

  Protocol for Saturday

1. Treat cells with EGCG or H2O2 accordingly.
2. At the end of treatment, aspirate off media and then add 200ul of lysis buffer to well.
3. freeze and thaw cells on dry ice or at -80*C three times, to ensure lysis. Vortex plates after each thaw
4. transfer lysates to 1.5ml tube for sonification. During transfer, pipet lysate and inject back into well to slough off cells from plate. Do this for 3 or 4x.
5. Sonicate lysates for ~4-5sec.
6. Spin down lysate for ~ 10 sec.
7 Remove ~ 100ul for luciferase assay. Use 30ul of luciferin.

Calculations
- catalase 6x3x3 = x72
- EGCG 6x3x2 = x36
- H2O2 6x3x2 = x36

- catalase 30u/ml 1.2x72 = 86.4 ul needed
- H2O2 10ul/well 360ul needed
- EGCG 25ul/well x36 = 900 ul  ¶ 10:17 AM

  Thursday (yesterday )
- 3pm removed tranfection mixture
- washed with 250ul/well PBS
- added 1ml/well media  ¶ 9:57 AM

  Wednesday (Yesterday )

- did a gel to see if there was DNA, came out positive
- used the spectrometer to measure DNA concentration

numbers were as expected, from comparing the thickness of the bands from the gel

---------------------------

Thursday (Today) - couldn't find transfection protocol, Dr. Chin wrote it up again

- Design for experiment
1. 3TP-LUX
2. 3TP-LUX Catalase
3. 3TP-LUX + EGCG
4. 3TP-LUX + EGCG + Catalase
5. 3TP-LUX + H2O2
6. 3TP-LUX + H2O2 + Catalase
* time points 0, 0.25, 0.50, 1, 2, 3 in triplicates


Protocol for transfection

1. take 216ul metafectene and add 10.8 of media to mix in 15ml tube
2. take 155ul of 3TP-LUX and add 16.2ml of media to mix in 50ml tube
3. then mix 1 + 2 together in the 50ml tube and use repeating pipettor to dispense 250ul of transfection mix into each well

10.8ml of media + metafectene
16.2ml of media + DNA

0.7ug/ul stock of 3TP-LUX 108/0.07=155ul

216ul of metafectene (2ul of metafectene/well)
108 ug of DNA (155ul of 3TP-LUX) (1ug of 3TP-LUX/well)
--------------
Today, Plated cells

Calcuations

108 wells needed ~ 120 wells
5 plates x24 well plates = 120 wells total

want to plate 20,000 cells/well
120 wells x 20,000 cells = 2.4x10^6 cells needed for 5 plates/120 wells
stock contained 1x10^6 cells/ml

2.4E6/1E6 = 2.4ml of stock needed to plate 120 wells
500ul/well needed x 120 wells = 60ml for 120 wells
2.4ml cells in 57.6 (60ml) of media
500ul of cell suspension with 500ul of media in each well to give 1ml of solution per well

use 0.5ml of stock and add to T75 flask  ¶ 10:26 AM

  1. Add 10ml of P1 buffer and ressuspend bacteria pellet throurouly

2. Add 10ml of buffer P2 (lysis buffer) and mix gently by inversion and then incubate at room temp for 5min
3. Add 10ml of buffer P3 (neutralization buffer) and mix gently by inversion
4. Pour lysate into Qiafilter cartridge and incubate at room temp for 10 min
5. White step 4 is on going , equilibirate Maxitip with 10ml of buffer QBT by gravity flow
6. Go back to Qiafilter in step 4 and use plunger to gently push lysate into Maxitip
7. Allow teh lysate to enter the column resin
8. Wash column with 60ml of buffer QC
9. Elute DNA with 15ml buffer QF into a 50ml tube
10. Precipitate DNA by adding 10.5ml (0.7 volume) of isopropanol and incubate at room temp for 5 min
11. attach syringe (30ml) to QIA precipitator and transfer mixture from step 10 into syringe and push throguh gently
12. dry QIA precipitator by pushign plunger through filter a second time
13. Elute with 1ml of TE buffer into microfuge tube
14. reelute by pouring the eluate from step 13 through QIA precipitator again  ¶ 11:39 AM

  Today
10:30 picked 2 colonies and pipetted them into 5ml of LB +amp  ¶ 1:33 PM

  Monday - split bes21 and performed transformation

1. Split the BES21 cells and resuspend in 6ml of media nad then take 3ml of cells and transfer to T75 flask
2. do a transformation with teh 3TP-LUX plastmid for another plasmid prep

Dilute the 0.7ug/ul stock of 3TP-Lux to 1ng/ul (take 1ul of 0.7ug/ul stock and dilute with 99ul of H2O).

Then take 5ul of the 1ng/ul stock for transformation into 50ul of bacteria on ice for ~ 10 min.

Then heat shock cells for 30 sec at 37*C and incubate on ice for 2 min.

Add 1ml of LB to cells and transfer to 10ml tube and place on shaker for 1hr at 37*C.

Take 50ul and spread on agar plate and incubate plate at 37*C overnight.  ¶ 10:25 AM

  Friday - luciferase assay for catalase, ecgc, h2o2

not what expected, inhibition
----------------
Sunday - came in to split cells, lab was locked, didn't know where to get bacteria to do transformation
----------------
Monday -

10AM began transformation to make more 3TP

11AM split cells  ¶ 10:56 AM

  yesterday - luciferase assay

3TP had mixed readings
GCCG - low

12AM Plated another 2 24-well plates

4PM washed and added 1ml media to each well

8PM
- added catalase
- added ECGC
- added H2O2


  ¶ 10:27 AM

  10AM lysed cells
- diluted 5x solution to 1x, 1ml in 10ml H2O
- did not wash cells
- added 200ul lysis solution

- vortexed
- frooze over dry ice
- defrosted in 37* incubator
- moved to 1.5ml vials

- sonification  ¶ 12:02 PM

  10:30AM Started transfection

2ul metafectene and 1ug of 3TP-Lux per well (x24 wells per plate)

2ul metafectene and 1ug of GCCG-LUX per well(x24 wells per plate)

each well added 200ul of media (100ul metafectene, 100ul DNA)

- used 15ml test tubes
- rounded 48 wells ~ 50
- 100ul media x 50 wells = 5ml media
- 50 x 2ul metafectene = 100ul metafectene in 5ml media for ~ 48 wells


3TP 0.7ug/ul

1ug * 1ul/0.7ug = 1.4ul

- 100ul media x 25 wells = 2.5ml media
- 1.4ul x 25 wells = 35ul in 2.5ml media for ~ 24 wells

GCCG 0.15ug/ul

1ug * 1ul/0.15ug = 6.6ul

- 6.6ul x 25 wells = 165ul in 2.5ml media for ~ 24 wells


2:30PM 4hrs later

aspirated transfection solution
washed with 200ul PBS per well
added 1ml media per well

splitted cells and plated another 2 plates (48 wells)

60% confluent cells in flask
resuspended to make 6ml stock
0.5ml in new flask
1ml in 50ml of media and then plated 1ml of solution per well


  ¶ 4:22 PM