Transcription and Splicing Protocol
Transcription reactions:
1) thaw 32P at RT
2) thaw reagents at 30C
3) make reaction mix – per each reaction
1 µL
1 µL
1 µL
0.2 µL
0.2 µL
0.5 µL
3.6 µL
2 µL
0.5 µL
10x T7 RNA Polymerase buffer
(contains spermadine, DTT, and MgCl2)
10x NTPs (4 mM A and C, 1 mM U and G)
10x 20 mM CAP/diguanosine
40 unit RNasin (to inhibit RNase activity)
0.1 M DTT (for active RNasin)
1 mg/mL linearized plasmid
ddH2O
0.00333 mM -UTP
add 2nd to last
T7 polymerase
add last
final concentration
1X buffer
0.4 mM T/C
0.1 mM U/G
2 mM GpppG
2 mM DTT
0.5 µg DNA
0.00067 mM -UTP
10 µL total
4) at 37C 1-4 hours (~2 hours)
5) pour polyacrylamide gel (0.8 mm 6%) and pre-run
6) add equal amount (10 µL) of formamide/EDTA stop dye to samples – save around 0.25 µL
for radioactivity count
st
7) load onto gel with 1 and last lanes containing dye only
8) run at 45C (25 Watts for 1 gel, 60 Watts for 2 gels)
9) after transcription reaction, pry open gel plates and sandwich gel between saran wrap
7) check count to determine film exposure time - 100x 5K+ 5 seconds
1-200 0.5’
10x
not enough radioactivity
8) expose gel to film and develop
9) tape gel to film and cut out gel band, discard gel
10) elute RNA from gel in 650 µL RNA elution buffer on rotator (can elute O/N)
11) split eluted RNA into 2 tubes with 300 µL each
12) add 750 µL EtOH for 10-30’ to precipitate RNA @-20C
13) store at -20C (can be stored up to 2 or 3 weeks in freezer – RNA degrades)
14) spin pre-mRNA at 14000 rpm 10’ to pellet
15) remove sup and air-dry
16) resuspend in ddH2O – amount depends on pellet cpm – ex. 600,000 counts into 75 µL
17) store @-20C
Splicing reactions:
1) make reaction mix
amounts for 1 reaction (* notes below)
final concentration
1
1 µL
1 µL
25 mM ATP (25x)
0.5 M Creatine Phosphate (25x)
(regulation of ATP, high energy
bond reconstitution system)
1 µL
80 mM MgCl2 (25x)
*1 µL
pre-mRNA
*0.25 µL
40 unit RNasin
(0.25 µL/25 µL reaction)
0.25 µL
100 mM DTT (100x)
5.77 µL
13% Polyvinyl alcohol
*
Nuclear extract from HeLa cells
*
2.5 M KCl
*
0.5 M Hepes pH 7.9
add ddH2O for a total of 25 µL for each reaction
*notes:
1 mM ATP
20 mM CP
3.2 mM MgCl
*
*10 units
1 mM DTT
*3% PVA
*10-50% NE
72.5 mM KCl
12 mM Hepes
add either pre-mRNA or NE last
negative control with t=0 by placing on dry ice after initiation of splicing
*pre-mRNA concentration is variable depending on radioactivity
*RNasin may not be necessary
*PVA may not be necessary
*KCl, Hepes, NE amounts have to be adjusted depending on expt
(NE is in 100 mM KCl and 20 mM Hepes (BC100))
(SR proteins are in BC 300)
BC100 = 100 mM KCl, 20 mM Hepes
BC300 = 300 mM KCl, 20 mM Hepes
BC850 = 850 mM KCl, 20 mM Hepes
Table for NE, KCl, Hepes amounts
%NE
NE
50
40
30
25
20
10
12.5 µL
10 µL
7.5 µL
6.25 µL
5 µL
2.5 µL
2.5M KCl
Total conc
72.5 mM
0.225 µL
0.225 µL
0.225 µL
0.225 µL
0.225 µL
0.225 µL
To determine SR protein stock molarity:
use concentration (mg/mL = g/L)
convert g to Da (1 Da = 1 g)
 (g/L) / (g/mol) = Molar
0.5M Hepes
Total conc
12 mM
0.1 µL
0.1 µL
0.1 µL
0.1 µL
0.1 µL
0.1 µL
BC100
0 µL
2.5 µL
5 µL
6.25 µL
7.5 µL
10 µL
ex. 0.4 mg/mL = 0.4 g/L
45 kDa prot = 45000 g (g/mol)
 (0.4 g/L) / (4500 g/mol)
2
= 9 x 10-6 M
= 9 µM
NE
Table for BC100, BC300, NE, and SR protein amounts
BC100
SR protein
BC300
0.5M Hepes
10 µL
1.5 µL
0.14 µL
2.5M KCl
0.15 µL
2) at 30C for 90’
3) make proteinase K digestion mix –
for each 25 µL reaction 100 µL
2x pK buffer
75 µL
ddH2O
2.5 µL
10 mg/mL proteinase K
1.5 µL
glycogen (ppt carrier)
4) add 175 µL of mix to each reaction
5) at 37C 12-15’
6) add 200 µL phenol:chloroform 1:1 to extract RNA
7) vortex 5-10 seconds
8) spin at 14000 rpm 2-5’
9) remove and save ~200 µL sup
10) add ~500 µL ethanol to ppt RNA
11) place at -20C for at least 10’
12) spin at 14000 rpm 10’ to pellet
13) remove sup
14) resuspend in 7 µL formamide/EDTA stop buffer
15) run on polyacrylamide gel (0.7 mm 6% and/or 10%
16) after gel run, can vacuum dry onto paper or sandwich gel between saran wrap
17) expose to film with intensifying screen at -80C O/N or expose to phosphor screen
3
For polyacrylamide gels:
1) clean plates with ethanol
2) set-up plates with clamps and spacers
3) make gel mix:
4) pour gel at angle
5) allow to polymerize
6) can pre-run to get rid of free radicals in Temed
For 5% polyacrylamide gels:
1x TBE 7 M Urea
20% polyacrylamide/bis 19:1 7 M
Urea 1x TBE
10% APS
Temed
20 mL
15 mL
5 mL
30 mL
22.5 mL
7.5 mL
40 mL
30 mL
10 mL
200 L
40 L
300 µL
60 µL
400 µL
80 µL
20 mL
14 mL
6 mL
30 mL
21 mL
9 mL
40 mL
28 mL
12 mL
200 L
40 L
300 µL
60 µL
400 µL
80 µL
20 mL
12 mL
8 mL
30 mL
18 mL
12 mL
40 mL
24 mL
16 mL
200 L
40 L
300 µL
60 µL
400 µL
80 µL
20 mL
10 mL
10 mL
30 mL
15 mL
15 mL
40 mL
20 mL
20 mL
200 L
40 L
300 µL
60 µL
400 µL
80 µL
For 6% polyacrylamide gels:
1x TBE 7 M Urea
20% polyacrylamide/bis 19:1 7 M
Urea 1x TBE
10% APS
Temed
For 8% polyacrylamide gels:
1x TBE 7 M Urea
20% polyacrylamide/bis 19:1 7 M
Urea 1x TBE
10% APS
Temed
For 10% polyacrylamide gels:
1x TBE 7 M Urea
20% polyacrylamide/bis 19:1 7 M
Urea 1x TBE
10% APS
Temed
4