I.  RNA probes for gel shift

 

A.  Reaction mixture

                                        Hot                                  Cold

5X buffer for T3 or T7      5                                      10         

200 mM DTT                   1                                      2           

dNTPs (4uM stock)                    2.5 (-UTP for hot)                     5            (10 nmol final)

32P-UTP (100 uCi final)     10                                    0           

RNAsin (40U)                  1                                      2           

lineraized DNA ~ 0.5 ug/ul     2                                      2           

T3 or T7 pol (20U)            1                                      2           

H2O                                 2.5                                   27         

                                        25                                    50

 

 

Template linearization and POL

 

                           A2A6 Hind III     T3

                           A2A6 Bam HI     T7

                           A3 Hind III         T3 (+)

                           A3 Not I             T7

 

incubate for 20 min at 37*C

stop rxn at 65*C for 5 min

 

B.  Add             

50 ul (hot) or 25 ul (cold) H20

             3 ul or 6 ul DNase I (20 u OR 40 u Final)

             1 ul tRNA-(10 mg/ml stock)

             incubate 37’ for 5 min

 

C.  Clean in vitro transcribed probe

                                                    

phenol extract: 1 vol                     79 ul

 

EtOH/ NaOAc ppt:        

total vol.                                         80

vol to add 3 M NaOAc                   8

vol to add 100% EtOH                    200

resuspend 100 ul in Munro’s reagent

 

D.  Pass through G-50 column (BMB G-50 spin column)

                  1.  Prepare the column by mixing the matrix.  You have to be forceful with the mixing.

2.  Open the top cap and then remove the bottom tip.  Allow the solution in the matrix to drip by gravity

3.  Place one of the supplied eppindorfs under the column and place the entire set-up into a 12 ml                         snap cap.

                  4.  Spin in beige clinifuge for 2 min at setting # 4 (1100 x g)

                  5.  Matrix should settle in an even tower.

6.  Apply the RNA (max vol. 100 ul)sample SLOWLY to the top of the Matrix -make sure the RNA does not run down the side of the column.

7.  Place a new eppi on to the bottom of the column and again place the set-up into a 12 ml snap cap.

                  8.  Spin for 4 min at setting # 4.

9.  Run a formamide agarose gel and blot to nitrocellulose (like a northern) to check integrity of RNA.  Formaldehyde agarose gel:  add 0.6-0.75 g agarose to 43.5 ml DEPC treated H20 in an RNase free flask. Melt. Add 5 mls 10X MOPS.  Cool to 50’C, add 2.55 mls 37% formaldehyde.  Pour.  Let sit 1 hour.  Flush wells. Load. Run in 1X MOPS.  Blot to Nitrocellulose under gel dryer with plenty of whatman to abs liquid (no heat-takes < 15 min).

 

II.  Preparation of protein extracts

 

A.  Mammalian cells:

                  1.  pellet of ~106 cells trypsinized-EDTA from tissue culture flask

                  2.  wash 2x in PBS

                  3.  lyse cells with 20 ul of Munro’s buffer + 0.2% NP-40

                  4.  keep on ice for 10 min

                  5.  add 80 uls of Munro’s buffer (W/O NP-40)

                  6.  pipette up and down 10X

                  7.  spin @ 14, 000 rpm/ 10 min in table top cold centrif.

                  8.  transfer SN to a clean eppindorf.

9.  measure protein concentration (this usually yields enough protein for about 5-10 binding reactions with 40 ug/rxn)

 

B.  T. cruzi (amastigotes or epimastigotes)

                  1.  pellet ~108 cells

                  2.  wash 2x in PBS

                  3.  lyse cells with 10 uls of Munro’s buffer + 0.1 % NP-40

                  4.  keep on ice for 10 min.

                  5.  add 180 uls of Munro’s buffer W/O NP-40

                  6.  homogenize using a 23 gauge needle and 1 cc syringe

                  7.  spin @ 14, 000 rpm for 10 min

                  8.  take SN and place in clean tube

                  9.  determine protein concentration

                  10.  store at -70*C if not used immediately

 

III.  Binding Reaction

 

             start pre-run of Native gel now (SDS PFGE needs no pre-run)

             1.  if sample requires cold or other competitor RNA add it to extracts before adding RNA probe

             2.  add 40 ug protein from appropriate cell extract (protein from about 80,000 cells)

3.  if extract is from T. cruzi increase KCl concentration to 200 mM-note Munro’s reagent has final salt of 40 mM.

             4.  add between 10,000-20,000 cpm hot RNA (about 0.1 ng RNA)

             5.  incubate 25 min @ RT

             6.  add RNase T1 and RNase A

             7.  incubate 10 min @ RT

             8.  add 1 ul  Heparin (stock @60 mg/ml) (to 5 mg/ml final concentration)

             9.  incubate 10 min @ RT

             10.  Cross link for 3500 (x100) uJ, 4 cm from source

11.  add approp loading buffer (for Native or SDS-page-(pre-boil required))

             12.  run @ 8 V/cm or 100 V for mini-gels or 85 V in stacking 130 V in seperating

Loading Buffer Native gel

                  30 mM Tris-HCl pH 7.5

                  40% sucrose (do not use glycerol buffer)

                  0.2 % bromophenolblue

 

2  6% Native Acrylamide Mini Gels

                  4 mls 30% Acrylamide/Bisacryl (29:1)

                  0.2 ml 10% APS

                  4 mls 5X TBE

                  16 ul TEMED

                  11.8 mls H20 (20 mls final-enough for at least 2 mini-gels)

                  running buffer is 1X TBE

 

2  10% Tris-glycine SDS polyacrylamide mini gels

5.0 mls 30% Acrylamide/Bisacryl (29:1)

                  5.9 mls H20

                  3.8 mls 1.5 M Tris pH 8.8

                  150 ul 10% SDS

                  150 ul 10% APS

                  6 ul TEMED

                  running buffer is 1x tris gyl buffer

 

2  5% Stacking Gels

0.83 mls 30% Acrylamide/Bisacryl (29:1)

                  3.4 mls H20

                  0.63 mls 1.0 M Tris pH 6.8

                  50 ul 10% SDS

                  50 ul 10% APS

                  5 ul TEMED

 

 

 

Munro’s binding buffer

                  10 mM HEPES, pH 7.6

                  3 mM MgCl2

                  40 mM KCl

                  5% glycerol

                  1 mM DTT

For lysis Buffer add 0.2% NP-40 (mammalian) or 0.1% NP-40 (t. cruzi)

 

 

rnabndshft-10/2/97


rnabndshft-7/17/97