Planning 1-26-12

2015-01-13

azim58 - Planning 1-26-12

How many more materials will I need to continue constructing my library?

I currently have about 1.3e6 colonies and I want 3e6 so I have 1.7e6
left. With my current transformation efficiency usually yielding around
4e4 colonies total in a tube I have about 1.7e6/4e4=42.5 transformations
left.

How many cuvettes, 96 well assay plates, 2 mL plates, Biomek tips, and
other materials will I need?

* 43 cuvettes
  o I only have 6 left. I can order more from Invitrogen with Cat no.
    P410-50
  o don't have enough

* Plates
  o How many wells will I have? 1.7e6/1e3=1.7e3. How many plates does
    this require? 1.7e3/96=17.7 plates
  o 96 well assay plates required=17.71*3=53.13 (the 3 is for
    overnight culture, 1/10 dilution, and glycerol stock)
    - don't have enough

o 96 well 2 mL plates required= 17.71 plates

* Tapesheets for covering plate
  o Airpore Tapesheets
    - 3 used per plate (1 used for overnight culture, 1 used for
      culture to 0.5 OD, 1 used after IPTG has been added)
    - so I need 3*18=54
    - definitely have enough

o 3M tapesheets
    - 2 per plate (1 for glycerol stock, 1 after lysozyme has been
      added)
    - so I need 2*18=36
    - definitely have enough

* Spectrophotometer cuvettes
* Electrocompetent cells: 43*20*uL= 860 uL
  o Have enough. I have 1000 uL

* Lysis Buffer
  o 100 uL needed per well. I need a total of 1,700*100*uL = 170,000
    uL lysis buffer plus I'll want a 30,000 uL buffer so 200,000 uL lysis
    buffer
  o 1X PBS: 9.85*mL*"PBS"/(10*mL*"solution")*200*ml*"solution" = 197
    mL PBS
    - have enough

o Triton X-100: 100*uL/(10*mL)*200*mL = 2 mL Triton-X 100
    - have enough

o 1 mM PMSF: 50*uL/(10*mL)*200*mL = 1 mL
    - pretty sure I have enough

o Protease inhibitor tablets : 1/(10*mL)*200*mL = 20 tablets
    - have enough

* Regular 200 uL tips (for removing supernatent) (X2)
  o If I use a new tip for each well.
    - would need 1700*2=3400 tips. How many tips come in a box?
      96*2 = 192. How many boxes would I need? 3400/192 = 17.71 boxes of
      tips

o If I reuse tips (I think this might be okay since the bacteria
    containing the protein are pelleted at the bottom and they are not
    lysed yet
    - I would only need 12*2=24 tips instead of 3400 tips. This
      sounds much better.

o have enough for sure

* LB/Agar Plates
  o Need 2 plates per electroporation. 2*43 = 86 plates

* IPTG
  o 11.1 uL needed per 100 uL culture. There is 1700*100uL = 170,000
    uL culture so I'll need 170,000*uL*11.1*uL/(100*uL) = 18,870 uL. I'll
    want a 30 mL buffer volume so the Biomek has enough liquid so I'll
    want a total of about 18,870*uL+30,000*uL= 48,870 uL 10 mM IPTG
  o How much powder is needed to make this much IPTG?
    10*mmol/(1*L)*1*L/(1*10^6uL)*48,870*uL*1*mol/(1000*mmol)*238.3*g/(1*mol
    ) = 0.1165 g IPTG.
  o have enough

* Lysozyme
  o 2.5*uL/100*uL*1700*100*uL = 4250*uL needed + 30,000*uL buffer
    34,250 uL of (10*mg/(5*mL) lysozyme
  o How much powder is needed. 10*mg/(5*mL)*34.25*mL = 68.5*mg
  o pretty sure I have enough

* Biomek Tips
  o Tips are used per sample plate or per batch depending on the
    step. In other words, for some steps a tip box is required for each
    sample step such as a step that transfers the LB bacteria solution to
    a new plate. On the other hand, some steps use the same tip box for
    multiple different sample plates since the tip does not touch any of
    the unique wells containing LB bacteria culture. An example of this
    can be seen in the step in which IPTG is added to multiple different
    plates with the same tip box.
  o I'll estimate that I have about 3 more batches required.
  o P250 Cat No 717252
    - 2 tips boxes per sample plate (1/10 dilution and glycerol
      stock, transfer to deep well plates and add lysis buffer) + x per
      batch (glycerol addition, LB addition, addition of IPTG, addition
      of DNAse and MgCl2)
    - So about how many more tip boxes will I need? 2*18+4*3 = 48.
      10 tip boxes come in one package of tip boxes. So I need 5 or 6
      packages of tip boxes

o P50 Cat No A21586
    - 2 tip boxes per batch (lysozyme addition, EDTA addition)
    - So I need about 2*3=6 tip boxes. I'll order 1 or 2 boxes of
      these P50s

* DNAse
  o 12.5 uL of 0.002 mg/mL per well
  o How many uL do I need? 12.5*uL/(1*well)*1700*well = 21250 uL So
    21.250 mL. How many mg needed? 0.002 mg/mL*21.250*mL = 0.0425 mg.
  o I definitely have enough

* MgCl2
  o I'm sure we have enough of this

* EDTA
  o I'm sure we have enough of this

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