Luciferase Assay

To make 1X Cell Lysis Buffer:

Note: This recipe is for 24 samples ONLY- please use accompanying chart for different sample sizes!!

(for 24 samples at 24-well-plate)

Lysis Stock (5X) 1540µl
Water 6160µl

(for 24 samples at 12-well-plate)

Lysis Stock (5X) 2980µl
Water 11920µl

Home made Cell Lysis Buffer (5X)

125µM Tris-PO4, 10µM DTT (dithiotretiol), 10µM CDTA (Sigma D0922), 50% Glycerol, 5% Triton x-100)

150ml

1M Tris-PO4 18.75ml
DTT 0.23g
CDTA 0.52g
Glycerol 75ml
Triton X-100 7.5ml
H2O 48.75ml

GME Buffer (1X)

(25mM Glycylglyine (Sigma G2265) , 15mM MgSO4, 4mM EGTA)

0.5M Glycylglycine 25ml
1M MgSO4 7.5ml
0.5M EGTA 11ml
H2O 456.5ml

ATP (Sigma A7699)

1g in 4.7ml H2O

100µl aliquots stored at -20ºC

Protocol

1)      Aspirate media from wells

2)      Wash wells with PBS briefly

3)      Add 400µl/well (12-well/plate) or 200µl/well (24-well/plate) 1X Cell Lysis buffer and lyse for 10-15min while occasionally shaking plate.

4)      Transfer supernatant to a fresh epi tube.

5)      Briefly spin at 13K for 30sec-1min to pellet debris.

6)      Transfer 200µl of supernatant (without contaminating debris) to glass tube for assay.

Note: This recipe is for 24 samples ONLY- please use accompanying chart for different sample sizes!!

Prepare Luciferase Subtrate Solution (Injector 1)

Luciferase Substate Stock 880µl
GME 3520µl

Prepare Mix Solution (Injector 2)

GME 8700µl
1M K3PO4 174µl
0.2M ATP 116µl
1M DTT 11.6µl

Flow Sorting for CSCs

Flow Sorting:

Separation/Isolation of ESA+ Human Breast Epithelial Cells

  1. Trypsinize cells (count)
  2. Spin down and wash in PBS
  3. Resuspend at 1×106 cells/ml in PBS++ (cold)

PBS++:  100ml PBS

1ml insulin

500 µl FCS

  1. Incubate cells with primary Ab 10 min at R.T.
  2. Wash by adding 4 ml PBS++ to incubating AbBCIC FACS protocol2.doc
  3. Spin and resuspend in ~300 µl minimum PBS++ (or appropriate volume).
  4. put samples on ice
  5. Bring tubes with FCS for collection.

Antibody amounts:

CD24:  20 µl/106 cells

CD44:  20 µl/106 cells

ESA: 10 µl/106 cells

Qiagen Miniprep Protocol

MiniprepProtocol.TIF

This protocol is designed for purification of up to 20 µg of high copy plasmid DNA from 1-5 ml overnight cultures of E. coli in LB (Luria-Bertoni) medium.

All protocol steps should be carried out at room temperature.

Procedure

1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.

Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.

If LyseBlue reagent has been added to Buffer P1, vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4-6 times.

Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

If LyseBlue las been added to Buffer P1 the cell suspension willt urn blue after addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.

3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.

To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.

If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue has gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.

4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.

A compact white pellet will form.

5. Apply the supernatant from step 4 to the QIAprep spin column by decanting or pipetting.

6. Centrifuge for 30-60 s. Discard the flow-through.

7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow-through.

This step is necessary to remove trace nuclease activity when using endA^+^ strains such as the JM series, HB101 and its derivatives, or any wildtype strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5α do not require this additional wash step.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30-60 s.

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.

Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 µl Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

DNA Quantification with Spectrophotometer in Charest Lab

Materials:

Protocol:

  1. turn on the spectrophotometer
  2. choose “dsDNA” and then “blank” from the touchscreen display
  3. wait for UV light to warm up
  4. while light is warming up, prepare a 1:20 dilution (5µl DNA + 95µl MilliQ water) in an Eppendorf tube
  5. gather up quartz cuvette (quartz cuvette will be located either in the drawer marked “stirbars markers razors tape” in the lab or in the top right drawer of the desk in room 14047), P200, and tips and return to the spectrophotometer
  6. on touchscreen display, change dilution to “20”
  7. re-blank using quartz cuvette filled with H2O
  8. fill cuvette with DNA dilution and put into spectrophotometer
  9. choose “read” on touchscreen
  10. write down “conc” (in µg/ml), “abs“, & 260/280 ratio

    260/280 ratio should be close to 1.80

  11. turn off spectrophotometer

See also: