User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/01

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Bradford, qPCR AKAP12 & AKAP9, Silverstaining Main project page
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Summary

Materials & Methods

Method

Bradford

  • Bradford performed by Tina
    • 5,05 mg/mL protein in Lysis buffer
    • 4,02 mg/mL protein in HeLa -S sample

SDS-PAGE

  • 8% SDS-PAGE
  • preparation of cDNA and qPCR
Gel loading hTERT D9 + HEK293 & HeLa-S see codes 21June2010
# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Gel
cAMP prec.
X X Invitrogen Marker
(8 μL)
HeLa -S
(20 μL)
HEK293
(20 μL)
Biorad Marker
(8 μL)
C1
(20 μL)
C2
(20 μL)
C3
(20 μL)
S1
(20 μL)
S2
(20 μL)
S3
(20 μL)
X X X
Coomassie staining
  • As described 25June2010
    • For silver staining fix again in 40% Ethanol/10% acetic acid (2x 10-30 min) & 10% EtOH/5% acetic acid (2x 10 min)
Silver staining
  • FIXING (see above when following Coomassie staining)

1. Fix gel for 2 h in fixing solution (30% ethanol (v/v) & 10% acetic acid (v/v))
2. Wash gel 2x 10 min. with 250 mL 10% 30 Ethanol (v/v)
3. Wash gel 3x 10 min. with dH2O 10 min.
OPTIONAL
a. Soak for 10 min in 3.4 mM potassium dichromate-0.0032 N nitric acid (200 ml/gel, 25°C) (stronger signal)
b. Wash with deionized water three times for 10 min (400 ml/gel, 25°C).
STAINING
4. Incubate gel 30 min. in 200 mL 0.1% Silvernitrate solution (w/v)
5. Rinse 3x 5 min. with dH2O
DEVELOPING
6. Develop gel for 5 – 10 min. in 200 mL 3% NaCO3 + 200 μL 37% Formaldehyde solution
7. Stop development with 200 mL 1~5% Acetic acid for 5~10 min.
8. Wash gel with dH2O (2 min.)
DESTAINING
9. Destain with (Farmer’s reducer, will clear whole gel!)
a. 50 mL 2% Potassium hexacyanoferrate(III) (C6N6FeK3)
b. 50 mL 3% sodiumthiosulphate (Na2S2O3)
10. Wash 3x 10 min. with dH2O
(RESTAINING)
(Drying the gel, gel can be soaked in drying solution (10% glycerol + 15% EtOH) for 30 min. and let to dry

qPCR

Preparing cDNA
  • SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen)
    • Incl. SuperScript® III Reverse Transcriptase (Invitrogen) (DRAWER #5)

The following procedure is designed to convert 1 pg to 5 μg of total RNA or 1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA:

  • Mix and briefly centrifuge each component before use.
  • Combine the following in a 0.2- or 0.5-ml tube:
Component Amount
up to 5 μg total RNA (1 μg)n μl
50 ng/μl random hexamers1 μl
Annealing buffer 1 μl
RNase/DNase-free water 8 μl
  • Incubate at 65°C for 5 min (progr. ALI), and then immediately place on ice for at least 1 minute. Collect the contents of the tube by brief centrifugation.
  • Add the following to the tube on ice:
    • 2X First-Strand Reaction Mix 10 μl
    • SuperScript™ III/RNaseOUT™ Enzyme Mix 2 μl
  • Vortex the sample briefly to mix, and collect by brief centrifugation (1 min. 1000rcf). Incubate as follows:
    • Random hexamer primed: 5-10 min at 25°C, followed by 50 min at 50°C (Progr. VANN)
  • Collect the reactions by brief centrifugation
  • cDNA synthesis reaction can be stored at -20°C or used for PCR immediately
Real time qPCR
  • Thermo Scientific Solaris qPCR Gene Expression Assay
    • Two primers at 800 nM + one probe 200 nM = primer probe set (20x)
  • Solaris qPCR Gene Expression Master Mix (2x) (ROX)
  • qPCR cycler (ABI StepOne)
  • MicroAmp™ Fast optical 48-well reaction plate (0.1 mL) (Applied Biosystems)
Primer
  • Primer AKAP9 (Dharmacon Inc.
    • FW: AACCTGAAGATGTGCCTCCTG
    • REV: CTGGAGTGCATACCTTTC
    • Probe: GATTTTGTCTAATGAAA
  • AKAP12 (Dharmacon Inc.
    • FW CAAGCACAGGAGGAGTTACAG
    • REV CTGGTCTTCCAAACAGACAATG
    • Probe ATCATGCAGTTAAACTCA
  1. Thaw reagents on ice, mix (DO NOT VORTEX) and spin down
  2. Mix
    • Solaris qPCR Master mix (2x) 12.5 μL
    • Solaris primer/probe set (20x) 1.25 μL
    • Water (PCR Grade) 9.25 μL
    • cDNA 2 μL
    • Total volume 25 μL
  1. Seal with optical clear seals and briefly centrifuge (removing bubbles)
  2. Run on thermal cycler (ABI StepOne)
' Temperature Time Number of cycles
Enzyme activation95 °C15 min1
Denaturation95 °C15 sec40
Annealing/Extension60 °C60 sec
  1. Data is collected during the last 40 cycles
48-wells plate
# 01 02 03 04 05 06 07 08
A S1 AKAP9 S1 AKAP9 S1 AKAP9 C1 AKAP9 C1 AKAP9 C1 AKAP9 NTC AKAP9 NTC AKAP12
B S2 AKAP9 S2 AKAP9 S2 AKAP9 C2 AKAP9 C2 AKAP9 C2 AKAP9 NTC AKAP9 NTC AKAP12
C S3 AKAP9 S3 AKAP9 S3 AKAP9 C3 AKAP9 C3 AKAP9 C3 AKAP9 NTC AKAP9 NTC AKAP12
D S1 AKAP12 S1 AKAP12 S1 AKAP12 C1 AKAP12 C1 AKAP12 C1 AKAP12
E S2 AKAP12 S2 AKAP12 S2 AKAP12 C2 AKAP12 C2 AKAP12 C2 AKAP12
F S3 AKAP12 S3 AKAP12 S3 AKAP12 C3 AKAP12 C3 AKAP12 C3 AKAP12

Notes

  • Dilution of RNA for preparation of cDNA
RNA H2O
S11,974,03
S22,423,58
S31,494,51
C11,644,36
C22,183,82
C31,574,43

Results

  • Coomassie staining

Discussion

  • Need a house hold gene to compare with

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Attachment

Bradford results, slightly different than those calculated by Tina

qPCR results, without GAPDH


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