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Molecular Biology DNA & RNA

Molecular Biology DNA, RNA & Miscell.

Ethanol precipitation of DNA

Plasmid reconstitution from filter paper

cAMP assay (Harden Lab)

Protein & Immunological Techniques

PKA assay (Corbin)

cAMP-agarose pulldowns

Antibody cross-linking to protein A/G with DMP

Purification of His6-PAK Column protocol

Purification of His6-PAK Batch/Column protocol (Talon Resin)

Expression & purification of PreScission GST fusion proteins

SDS-PAGE BioRad MiniGels

Western blotting

Stripping western blots

Lysis of cultured cells General protocol

Lysis buffer recipes

Immunoprecipitation general protocol

Immunoprecipitation Kinase Assay (IPKA) MAPK

IPKA HA-tagged MAPK (alternate protocol for MAPK IPKA)

IPKA MEK

IPKA PAK

IPKA c-Abl

In-gel kinase assay #1

In-gel kinase assay #2

TCA precipitation of proteins

Coomassie staining of gels for Mass Spec

Bacterial Culture

Transformation of competent cells

Cell Culture

Suspension culture

PC12 cell culture

Transfection of fibroblasts SuperFect

Transfection of NIH3T3 cells LipfectAMINE

Preparation of Vitrogen collagen gels (from Cohesion Tech)

Preparation of fibroblast-embedded collagen gels

Immunofluorescence & Microscopy

Coverslip preparation Clean, Cleaner, and For Cryin Out Loud

General immunofluorescent staining Formaldehyde fix

Phalloidin staining Long protocol

Fluorescent staining Actin cytoskeleton and Nuclei (short protocols)

Microtubule fixation & immunofluorescence

Immunofluorescent staining of pseudopod preps

DAPI staining Transwell inserts (Migration Assay)

Inverse Transwell plating technique

Appendix

Some Common Solutions

 


1             Molecular Biology DNA, RNA & Miscellaneous

Ethanol Precipitation of DNA in solution

 

1) Add 1/10th the volume (of the DNA solution) of 3M Na Acetate, pH 5.2 (40 l to 400 l).

2) Add 2.5 volumes of absolute ethanol.

3) Precipitate DNA -70 C for 30 min or -20 C overnight.

4) Pellet DNA, 14,000 rpm at 4 C for 30 min.

5) Decant supernatant, add 1 ml 70 % EtOH (precooled to -20 C). (This step washes away co-precipitating salts)

6) Centrifuge 14,000 rpm (~16,000xg) 10 min.

7) Decant supernatent, dry pellet, resuspend in T.E./ddH2O at 65 C.

<TOP>

 

Reconstitution of plasmids shipped on filter paper May 1, 2003

 

(Adapted from Guthrie.org)

 

1) Cut the filter paper into pieces that easily fit into a 1.5 ml microcentrifuge tube.

2) Add 100 l of nanopure water or TE buffer (10 mM Tris base, 1 mM EDTA, pH 8.0) to the microcentrifuge tube, vortex briefly, incubate at room temperature for 5 minutes, and repeat the vortex.

3) Centrifuge the tube for a few seconds and then remove 1-2 l of supernatant for use in transformation of E. coli.

<TOP>

 

cAMP assay Harden Lab (Shelley Hooks) August 2002

 

1) Transfect 50% confluent cells (COS-7) in 12-well plates 24-48 hours prior to assay.

2) Label cells with 3H-adenine (ART-143 Adenine [8-3H] 1mCi/mL, 20 Ci/mmol (American Radiochemicals, St. Louis)). Dilute 3H-adenine 1:100 in media (DMEM + 25 mM Hepes). Aspirate media from cells and add 500l of 3H media (5mCi/well). Incubate at 37 C for 2 hours.

3) While cells are labeling, Regenerate columns (1 mL bed volume):

Successively wash Dowex columns with

10 ml H2O

5 ml 1 M NaOH

10 ml H2O

5 ml 1 M HCl

2 X 10 ml H2O

Successively wash Alumina columns with

2 x 10 ml 50mM Tris

4) Treat cells:

- Add IBMX (200 M final) (50 l /well of 2.4 mM in DMEM + 25 mM Hepes) and incubate 10 min at 37oC

- Add 50l /well of 12X agonist (in DMEM + 25 mM Hepes) & incubate 15min at 37oC

- Aspirate and add 900l cold 5% TCA and incubate 10 min on ice

5) Separate cAMP and ATP on columns

- Load liquid from each well onto a Dowex column and allow to flow into basin.

- Once columns are dry, transfer column rack onto scintillation vials, and elute ATP into vials with 3mL H2O per column. Add scintillant to vials, cap and count for 3H ATP quantitation.

- Place rack of Dowex columns on top of Alumina columns, so that Dowex eluate is loaded onto alumina.

- Add 3mL H20 into each Dowex column, and allow eluate to flow into alumina column; allow alumina eluate to flow into basin.

- Once liquid has penetrated both columns, transfer Alumina columns onto scintillation vials and elute 3H cAMP with 4 mL 50 mM Tris. Add scintillant to vials, cap and count.

<TOP>

 

 

2             Protein & Immunological Techniques

PKA Activity Assay Cell Extracts

(adapted from Corbin & Reimann (1974) Methods Enzymol. 38:287-294)

 

1) Wash cells (e.g. in a 10cm plate) twice with ice-cold PBS and once with ice-cold buffer H (50mM b-glycerophosphate (pH 7.4), 1.5mM EGTA, 1mM DTT)

2) Scrape cells into 1ml buffer H (per 10cm plate or equivalent) & sonicate for two 15s bursts, using a Branson Sonifier set at 7 (or equivalent). Keep samples as cold as possible during & after sonication.

3) Spin lysates at 16000xg (i.e. 14000rpm in an Eppendorf microcentrifuge) at 4o C for 10min & reserve supernatant, taking care not to carry over any insoluble material. You will need much less than 1ml, so the pellet should be easily avoided.

 

(Note: Some cell lysates are better cleared at this speed than others. We have obtained excellent results on samples after ultracentrifugation at 50000-75000xg for 30min. Also, you may want to reserve the pellet for western blot analysis of residual PKA catalytic subunit. For this, wash the pellet 2-3 times with cold buffer H, add ~500l of 1X sample buffer, sonicate or vortex vigorously, boil for 5min, spin at 14000rpm in a microfuge, and run 20-30l of the supernatant for western blotting.)

 

4) a. For each sample or lysate, you will set up 4 reactions; -/+ Kemptide, -/+ PKI

Kemptide = LRRASLG (MW=772Da), 10mg/ml, Promega

PKI = TTYADFIASGRTGRRNAIHD (MW=2221Da), 10mg/ml, Promega

b. 3x PKA Buffer= 75mM b-glycerophosphate pH 7.4

3.75mM EGTA

30mM MgCl2

1.5mM DTT

c. For each sample, set up the following four tubes:

 

Tube A

Tube B

Tube C

Tube D

3X PKA Buffer

10l

10l

10l

10l

Kemptide (10mg/ml)

-

0.3l

-

0.3l

PKI (10mg/ml)

-

-

0.2l

0.2l

32P-ATP (10mCi/ml)

0.2l

0.2l

0.2l

0.2l

H2O (to 20l final volume)

9.8l

9.5l

9.6l

9.3l

 

 

 

 

 

 

 

 

It is obviously best to make up all the reaction mix you need for all the individual reactions, then aliquot 20l into the appropriate tubes.

d. Add 10l of extract to each tube, for a total reaction volume of 30l (20l mix+10l extract)

5) Incubate the reactions for 10min at 30oC or 30min at RT.

6) Spot 20l of each reaction onto pre-cut and pre-labeled squares (~ in x in) of P81 phosphocellulose paper (bulk paper available from many sources; pre-cut and number squares available from UBI and Pierce) and wash three times (~5min each) in a large volume of 150mM (~1%) phosphoric acid (10mls concentrated acid /L H2O).

 

(For this, we use a home-grown wash unit consisting of a large glass beaker into which fits a smaller plastic beaker with a wide-brimmed, triangular top. The plastic beaker has several small holes (about the size of the top of a disposable Pasteur pipette) poked in the bottom and around the sides near the bottom. Place a stir bar and the plastic beaker in the glass beaker, fill with wash buffer, and place atop a stir plate, set at a moderately high speed. After spotting the reaction onto the paper, drop the paper into the plastic beaker. To change the buffer, slowly & carefully withdraw the plastic beaker from the glass beake