Recipes and Reagents
Enzyme used for probe amplification is Platinum Pfx DNA polymerase (Invitrogen)
B213 (biotinylated control oligo): 5’-B-CTGAACGGTAGCATCTTGAC-3’
Prepare 5nM B213 solution by serial dilutions:
- Dissolve dry oligo at 100µM and dilute 1:100 (10µl 100µM primer in 990µl sterile 10mM Tris pH7.5), mix very well to obtain 1µM.
- Do another 1:100 dilution (10µl of 1uM primer in 990µl sterile 10mM Tris pH7.5), mix very well to obtain 10nM.
- Dilute 1:2 (100µl 10nM in 100µl sterile 10mM Tris pH7.5) to obtain 5nM.
PCR_B-fw: 5’-B-AATGGACTATCATATGCTTACCGTAACTTGAA-3’
PCR_rev: 5’-TGTGGATGAATACTGCCATTTGTCTCGAGGTC-3’
Prepare mixed stock of PCR_B-fw and PCR_rev, each at 20µM in sterile 10mM Tris pH7.5, and split into aliquots for frozen storage.
Blocking oligos:
Block_1 5’-AATGGACTATCATATGCTTACCGTAACTTGAA-3’
Block_2 5’-TTACCTGATAGTATACGAATGGCATTGAACTT-3’
Block_3 5’-GTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG-3’
Block_4 5’-CGGTGTTTCGTCCTTTCCACAAGATATATAAAGCCAAGAAATCGAAATAC-3’
To prepare 1000µl of blocking oligo solution with each oligo at a final concentration of 20µM:
- Dissolve dry oligos at 200µM in sterile 10mM Tris pH7.5.
- Add 100µl of each oligo stock to 600µl of sterile 10mM Tris pH7.5, mix well.
WASH A (Non-stringent wash buffer):
20XSSPE 300ml
10% Tween 1ml
Milli-Q water 699m l
1000ml
WASH B (Stringent wash buffer):
12x MES stock 83.3ml
5M NaCl 5.2ml
10% Tween20 1ml
Milli-Q water 910.5ml
1000ml
Filter Wash B and store at 4°C wrapped in foil.
12X MES
MES free acid monohydrate 7.04 g
MES Sodium Salt 19.3 g
Molecular Biology Grade water 80 ml
Mix well for >10 min on stirrer, pH to 6.5-6.7,
Adjust volume to 100ml.
Pass through a 0.2µm filter and store at 4°C, shielded from light.
Solution can be stored up to one year. Changes colour when it goes bad.
2X Hybridization Buffer
12X MES Stock 8.3 ml
5M NaCl 17.7 ml
0.5M EDTA 4.0 ml
10% Tween 20 0.1 ml
Milli-Q water 19.9 ml
50.0ml
Pass through a 0.2µm filter and store at 4°C, shielded from light.
Solution can be stored up to one year. Changes colour when it goes bad.
2X MES stain buffer
12X MES Stock 8.3 ml
5M NaCl 18.5 ml
10% Tween 20 1 ml
Milli-Q water 22.2ml
50.0ml
Pass through a 0.2µm filter and store at 4°C, shielded from light.
Solution can be stored up to one year. Changes colour when it is bad.
IgG antibody
Dissolve 30mg IgG/ml in sterile, filtered 150mM NaCl. OK to heat to 55°C and vortex slightly.
Mix well, looks blurry but OK.
Make 25µl-aliqouts, and store at 4°C.
Biotinylated anti-streptavidin phycoerythricin preparation ("BAS")
Reconstitute by adding 1ml Molecular Biology Grade water to 0.5mg antibody.
Mix well and make 25ul-aliqouts. Store at 4°C.
BSA
Dissolve 50mg Bovine Serum Albumin/ml in Molecular Biology Grade water.
Filter-sterilize and aliquot (make 1ml and 50ul-aliqouts). Store at -20°C.