User:TChan

• Project Overview
• Schematics
• Oligo List
• Scaffold
• Images
• iGEM Notes

RhBLCL Protocol

ISSUES

• Triplicate or not (ie. pool 12 wells, or pool 4 wells ?

• Do we need more than one uninfected "none" per cell line? Should be no difference between the timepoints

• Max volume is 250ul, but we might not want to subtract any volume, instead just adding to it three times (if we don't feel removal of previous reagents is necessary), so:
  • How much siRNA+transfection/R15 (for the "none" category) reagents to add?
  • How much LPS/IL-6+IL-6SR/R15 (for the "siRNA only" category) to add?
  • How much HIV GFP to add?

• Will we use only 1 siRNA (will have an siRNA reagent control for each timepoint plate of each cell line)?

• Should we also do this experiment for cells without siRNA (ie.another plate)?

• What is the treatment conc. of LPS for in vitro BLCLs?

• Do the IL-6 and SR tubes contain 10ug?

• Should we add to total volume of 150ul with the IL-6 and IL-6SR, and then 1-12hrs later remove 100ul and add 50ul virus + 150ul R15 for a total volume of 250ul during infection, as we normally do?

• Should we use 50ul SIV GFP, or a smaller amount, since we're only doing one viral conc.?

SCHEDULE

[W 5/14/08 8AM] (-) PREP CELLS, FORMULATE MIXES, and FORMULATE siRNA REAGENTS

• Spin down, resuspend in R15 no P/S, and count

• Plate cells into three (?) plates (one for each working CTR9-siRNA, and one for just transfection reagent control?) of each cell line at 10,000 cells/well (for siRNA protocol, will be plating 100ul of cells/well, so set every cell line to 0.3e6/ml before plating)

• Formulate LPS, IL6 and IL6SR using, resp., 1ml R15 no P/S, 1ml 2%FBS (assuming IL-6 tube contains 10ug), and 1ml 2%FBS

• Mix siRNA transfection reagents and time it so that transfection can happen ~ (0hr)

[W 9AM] (0hr) siRNA TREAT ALL PLATES

• siRNA treat/transfection reagent treat all five time point plates of each cell line:
  • Use only one siRNA
  • Mix up appropriate amounts of transfection reagents, incubate, add siRNA+reagents
  • Add 50ul to each well

[Th 9AM] (24hr) LPS/IL-6 TREAT "3hr" "6hr" "12hr"

• Remove 100ul from each well of the "3hr," "6hr," and "12hr" plates
• Pour 10mL of R15 no P/S into a trough
  • (100ul/well * 12 wells * 2 infection types * 3 plates/cell line = 7.2mL)

• Add 100ul of R15 no P/S to the "none" and "siRNA" rows

• Make up a solution of 20ul of each IL-6 and IL6SR, in 20ml R15 no P/S
  • Treatment strength is 1ul/mL, and will be in addition to 50ul already in well, so need 0.15ul/well of total volume 150ul = 0.15ul/100ul of solution to be added = 1.5ul/ml of the solution to be added
  • Thus, make up a solution of 20mL R15 no P/S with 30uL of IL-6 and 30uL IL-6SR
    • (100ul/well * 12 wells * 2 infection types * 3 plates/cell line * 2 cell lines = 14.4ml)

• IL-6+IL-6SR treat the two rows of each plate of each cell line from the "3hr," "6hr," and "12hr" plates by adding 100ul/well
• Make up a solution of 20ul of LPS in 20ml R15 no P/S (conc. to be determined)
• LPS treat the two rows of each plate of each cell line from the "3hr," "6hr," and "12hr" plates

[Th 10AM] (25) LPS/IL-6 TREAT "2hr"

• Remove 100ul from each well of the "2hr" plates
• Pour out 6ml R15 no P/S into a trench
• Add 100ul of R15 no P/S to the "none" and "siRNA" rows (100ul/well * 8 wells * 2 infection types * 3 plates/cell line = 4.8mL)
• In another trench, mix solution of 20ml R15 no P/S, 20ul of IL-6 stock, and 20ul of IL-6SR (treatment strength is 1ul/mL, and 100ul/well * 8 wells * 2 infection types * 1 plate/cell line * 2 cell lines = 3.2ml)
• Add 100ul of IL-6+IL-6SR solution to the two rows of each plate of each cell line from the "2hr" plates
• LPS treat the two rows of each plate of each cell line from the "2hr" plates: formulation to be determined

[Th 11AM] (26) LPS/IL-6 TREAT "1hr"

• Remove 100ul from each well of the "1hr" plates
• Pour out 6ml R15 no P/S into a trench
• Add 100ul of R15 no P/S to the "none" and "siRNA" rows (100ul/well * 8 wells * 2 infection types * 3 plates/cell line = 4.8mL)
• In another trench, mix solution of 20ml R15 no P/S, 20ul of IL-6 stock, and 20ul of IL-6SR (treatment strength is 1ul/mL, and 100ul/well * 8 wells * 2 infection types * 1 plate/cell line * 2 cell lines = 3.2ml)
• Add 100ul of IL-6+IL-6SR solution to the two rows of each plate of each cell line from the "1hr" plates
• LPS treat the two rows of each plate of each cell line from the "1hr" plates: formulation to be determined

[Th 12PM] (27) INFECT "1hr," "2hr," and "3hr" PLATES

• Thaw 11 tubes of SIV GFP (50ul/well * 8 wells/row * 4 rows/plate * 3 plates/cell line at this time * 2 cell lines = 9.6mL)
• Remove 100ul from each well of the "1hr," "2hr," and "3hr" plates
• Add back 150ul of R15 no P/S to the "infected" side and 200ul of R15 no P/S to the "uninfected" side
• Add 50ul SIV GFP to each of the "infected" side's wells
• Spin for 2hrs at 2400RPM

[Th 2PM] (29) REMOVE "1hr," "2hr," and "3hr" PLATES FROM CENTRIFUGE AND COLLECT CELLS FOR MICROARRAY RNA

• Pool "uninfected" cells into triplicate RNAse-free microcentrifuge tubes (4 wells/tube; 4 treatments in the uninfected side * 3 tubes/treatment * 3 timepoint plates * 2 cell lines = 72 tubes)

[Th 3PM] (30) INFECT "6hr" PLATE

• Thaw 4 tubes of SIV GFP (50ul/well * 8 wells/row * 4 rows/plate * 1 plates/cell line at this time * 2 cell lines = 3.2mL)
• Remove 100ul from each well of the "2hr" plate
• Add back 150ul of R15 no P/S to the "infected" side and 200ul of R15 no P/S to the "uninfected" side
• Add 50ul SIV GFP to each of the "infected" side's wells
• Spin for 2hrs at 2400RPM

[Th 5PM] (32) REMOVE "6hr" PLATES FROM CENTRIFUGE AND COLLECT CELLS FOR MICROARRAY RNA

• Pool "uninfected" cells into triplicate RNAse-free microcentrifuge tubes (4 wells/tube; 4 treatments in the uninfected side * 3 tubes/treatment * 1 timepoint plate * 2 cell lines = 24 tubes)

[Th 9PM] (36) INFECT "12hr" PLATE

• Thaw 4 tubes of SIV GFP
• Remove 100ul from each well of the "1hr" plate
• Add back 150ul of R15 no P/S to the "infected" side and 200ul of R15 no P/S to the "uninfected" side
• Add 50ul SIV GFP to each of the "infected" side's wells
• Spin for 2hrs at 2400RPM

[Th 11PM] (38) REMOVE "12hr" PLATES FROM CENTRIFUGE AND COLLECT CELLS FOR MICROARRAY RNA

• Pool "uninfected" cells into triplicate RNAse-free microcentrifuge tubes (4 wells/tube; 4 treatments in the uninfected side * 3 tubes/treatment * 1 timepoint plate * 2 cell lines = 24 tubes)

[Sat 12PM - 9PM] (~75 - 84) FIX AND FLOW THE "INFECTED" SIDES of ALL 10 PLATES

• 8 wells/treatment * 4 treatments * 5 timepoints * 2 cell lines = 320 wells total to flow

Notebook

<calendar> name=TChan/Notebook date=2007/2/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

<calendar> name=TChan/Notebook date=2007/5/01 view=threemonths format=%name/%year-%month-%day weekstart=7 </calendar>

Contact Info

 Tiffany Chan
 Biochemical Sciences
 Kirkland '07
 Academic Interests: DNA nanostructures, non-coding RNAs (SO. HOT.), drug design and delivery
 chan.tiffany at gmail.com
 Currently taking Biophysics 101