APPENDIX
A.1 CaCl2 Solution for transformation
60 mM CaCl2
15% (v/v) glycerol
10 mM PIPES, pH 7.0
Dissolve 1.47 g of CaCl2, 0.302 g of PIPES in 60 ml of dH2O. Add 15 ml of glycerol
and adjust the pH to 7.0. Bring the volume to 100 ml with dH2O. Filter sterilize using
a disposable filter unit, or autoclave. Store at 4oC.
A.2 Buffers for Plasmid DNA Isolation
A.2.1 Bacterial Resuspension solution (BRS)
50 mM glucose
50 mM Tris-Cl, pH 8.0
10 mM EDTA
Filter-sterilize and store at 4oC.
A.2.2 Lysis Solution
200mM NaOH
1% SDS
Autoclave and store at RT.
A.2.3 Neutralization Solution (KoAc)
3M potassium/5M Acetate
For 100 ml, take 29.4 g of potassium acetate, add dH2O to 88.5 ml, and 11.5 ml of
glacial acetic acid. Store at RT.
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A.2.4 Buffer P1 (resuspension buffer)
50 mM Tris·Cl, pH 8.0
10 mM EDTA
100 μg/ml RNase A
Store 2-8°C
Dissolve 6.06 g Tris base, 3.72 g Na2EDTA.2H2O in 800 ml dH2O. Adjust the pH to
8.0 with HCl. Adjust the volume to 1 liter with dH2O. Add 100 mg RNase A per liter
of P1.
A.2.5 Buffer P2 (lysis buffer)
200 mM NaOH
1% SDS (w/v)
Store 15-25°C
Dissolve 8.0 g NaOH pellets in 950 ml dH2O, 50 ml 20% SDS (w/v) solution.The
final volume should be 1 liter.
A.2.6 Buffer P3 (neutralization buffer)
3.0 M potassium acetate, pH 5.5
Store 2-8°C
Dissolve 294.5 g potassium acetate in 500 ml dH2O. Adjust the pH 5.5 with
glacial acetic acid (~110 ml). Adjust the volume to 1 liter with H2O.
A.2.7 Buffer QBT (equilibration buffer)
750 mM NaCl
50 mM MOPS, pH 7.0
15% isopropanol (v/v)
0.15% Triton X-100 (v/v)
Dissolve 43.83 g NaCl, 10.46 g MOPS (free acid) in 800 ml dH2O. Adjust the pH
to 7.0 with NaOH. Add 150 ml isopropanol and 15 ml 10% Triton X-100 solution
(v/v). Adjust the volume to 1 liter with H2O.
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A.2.8 Buffer QC (wash buffer)
1.0 M NaCl
50 mM MOPS, pH 7.0
15% isopropanol (v/v)
Dissolve 58.44 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O. Adjust the
pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter
with dH2O.
A.2.9 Buffer QF (elution buffer)
1.25 M NaCl
50 mM Tris·Cl, pH 8.5
15% isopropanol (v/v)
Dissolve 73.05 g NaCl and 6.06 g Tris base in 800 ml dH2O and adjust the pH to 8.5
with HCl. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with dH2O.
A.2.10 Buffer QN (elution buffer)
1.6 M NaCl
50 mM MOPS, pH 7.0
15% isopropanol (v/v)
Dissolve 93.50 g NaCl and 10.46 g MOPS (free acid) in 800 ml dH2O and adjust the
pH to 7.0 with NaOH. Add 150 ml pure isopropanol. Adjust the volume to 1 liter with
dH2O.
A.2.11 Buffer TE
10 mM Tris-HCl, pH 8.0
1 mM EDTA
Dissolve 0.157 g of Tris-HCl and 0.037 g of Na2EDTA.2H2O in 80 ml of dH2O.
Adjust the pH to 8.0 and bring the volume to 100 ml with dH2O.
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A.3 Antibiotic Solutions
A.3.1 Ampicillin (Stock: 10 mg/ml in dH2O; Working conc: 75 μg/ml)
Soluble in H2O and stored at 2-8oC.
Interferes with the final stage of synthesis of bacterial cell wall
A.3.2 Chloramphenicol 20 mg/ml in 80% ethanol (Working conc.: 5 μg/ml)
Inhibits elongation at peptidyl transferase
Soluble in H2O + NaOH, Stored at RT
A.3.3 Kanamycin (10 mg/ml in dH2O) (Working conc.: 100 μg/ml)
Soluble in H2O and stored at 2-8oC.
Interferes with bacterial protein synthesis by binding to 30S subunit of ribosomes
Stored at -20°C
A.4 Lysis Buffer for Genomic DNA Extraction
50 mM Tris-HCl, pH 8.0
50mM EDTA
1% SDS
10mM NaCl
A.5 6X Loading Dye for agarose gel electrophoresis
10 mM Tris-HCl (pH 7.6)
0.03% bromophenol blue
0.03% xylene cyanol FF
60% glycerol
60 mM EDTA.
In 1% agarose gels bromophenol blue co-migrates with ~300 bp DNA, while
xylene cyanol FF co-migrates with ~4000 bp DNA.
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A.6 0.5M Ethylenediaminetetra-acetate disodium salt (EDTA)
Add 186.1g of disodium ethylenediaminetetra-acetate 2H2O to 800 ml of H2O. Stir
vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~ 20g of NaOH
pellets). Dispense into aliquots and sterilize by autoclaving.
A.7 Ethidium Bromide (10 mg/ml)
Add 100 mg of Ethidium bromide to 10 ml of H2O. Stir on a magnetic stirrer for
several hours to ensure that the dye has dissolved. Transfer the solution to a dark
bottle and store at RT. Ethidium bromide is a powerful mutagen and is moderately
toxic. Gloves should be worn while working with ethidium bromide solution.
A.8 Restriction digestion of c-Kit plasmid (pcDNA3-c-Kit) with BamHI enzyme
i. pcDNA3-c-Kit plasmid
ii. Restriction enzyme BamHI
iii. 10X buffer for enzyme
iv. TE Buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.
A.9 Isolation of digested c-Kit fragment and pTre2Hyg digested fragment from
gel (QIAquick Gel Extraction Kit)
i. Gel slices containing c-Kit insert and linearized pTre2Hyg plasmid
ii. 1.5 ml Microcentrifuge tubes
iii. QIAGEN spin columns
iv. Buffer QG and Buffer PE
v. Microcentrifuge
vi. TE buffer (10 mM Tris pH=8.0-8.5; 1 mM EDTA)
A.10 LB Agar plates with antibiotic (1L)
15 g of agar was added to 1 liter of LB medium in a conical flask. The pH was
adjusted to 7.0 with 1N NaOH and autoclaved. The medium was allowed to cool to
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55°C before adding antibiotic (Ampicillin, 75 μg/ml final concentration;
Chloramphenicol, 50 μg/ml final concentration). 20-25ml of medium was poured into
each 75 mm petri dish and allowed to set.
A.11 Detection of c-Kit DNA in U937 cell line (which lacks endogenous c-Kit
expression) by polymerase chain reaction
i. Oligonucleotide primers complementary to the 5‘ and 3‘ ends of sequence to be
amplified (Integrated DNA Technologies, USA), stored at -20 C.
ii. Sterile dH2O (UV-sterilized for 5 min at 254-300 nm).
iii. PCR stock solutions, dedicated to PCR use only (Filter-sterilized and autoclaved).
1M Tris-HCl pH 8.3
1M KCl
1M MgCl2
iv. 10X PCR buffer (Bangalore Genei, India)
100 mM Tris-HCl pH 8.3
500 mM KCl
15 mM MgCl2
v. 10mM dNTPs stock solution containing 2.5 mM each of dATP, dTTP, dGTP and
dCTP.
vi. Taq DNA polymerase (Bangalore Genei, India), 1 U/0.34 l
vii. Template cDNA in plasmid vector (10 ng/ l)
A.12 Characterization of amplified Tet-off system plasmids
i. Tet-off system plasmids
ii. Microcentrifuge tubes (1.5 ml)
iii. Enzymes
iv. 10X buffer for enzymes
v. Sterile dH2O
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A.13 Amplification of c-Kit plasmid (pcDNA3-c-Kit) by large scale plasmid
DNA preparations (Maxiprep)
i. 500 ml sterile conical flasks.
ii. 50 ml autoclaved polypropylene centrifugation tubes, and suitable rotor and
centrifuge.
iii. Terrific Broth (TB) medium.
iv. Autopipettes of 20, 200, and 1000 l
v. BRS (Bacterial resuspension solution)
vi. LS (Lysis solution)
vii. KoAc (Neutralization solution)
viii. Isopropanol
ix.70% ethanol
x. RNase A. 10 mg/ml stock was prepared and stored at -20 C.
xi. TE buffer: 50 mM Tris-HCl (pH 8.0), 10mM EDTA
A.14 Amplification of pTet-off and pTre2Hyg plasmids using Qiagen endofree
plasmid Maxi kit
i. Inoculation loop
ii. Bacterial culture tubes (5 ml) and flasks (500 ml)
iii. 37°C Incubator Shaker
iv. Refrigerated centrifuge
v. Polypropylene centrifugation tubes (50 ml)
vi. QIAGEN-tips (Tip-2500)
vii. Isopropanol
viii. 70% Ethanol
ix. Tris-EDTA buffer
----End----
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CONFERENCES
1. Participated in 1st Annual Meeting of Stem Cell Research Forum of India
(SCRFI) and International Conference on Stem Cell Research, held at
Bangalore from 29th January to 1st February 2007
2. Attended symposium on ―Molecular Medicine: The Emerging Frontiers‖ and
delivered a talk on ―Stem cells: Proliferation control using c-Kit and HOXB4
genes‖ held at Maharshi Dayanand University (MDU), Rohtak, Haryana on
9thJan 2008.
3. Flow cytometry and its applications. 18th-21st Dec 2001, held at Defence
Institute of Nuclear Medicine and Allied Sciences, Lucknow Road, Timarpur,
Delhi-110054.
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RESEARCH PUBLICATIONS
1. Stem cell c-Kit and HOXB4 genes: critical roles and mechanisms in selfrenewal, proliferation, and differentiation. Sharma et al; 2006, Stem Cells
Dev. (USA) 15(6):755-78.
2. Stem cell factor receptor (c-Kit) induces clusters of genes regulating
pleiotropic functions, proliferation, differentiation, adhesion and survival
as revealed by high throughput transcriptome profiling. Cell Proliferation.
(Under Review) Sharma et al; 2011.
3. Mesenchymal stem cell-based therapy: a new paradigm in regenerative
medicine. Sharma et al., 2009. J Cell Mol Med., 13(11-12):4385-402.
4. Mesenchymal stem cells: molecular targets for tissue engineering. Sharma
et al., 2007. Stem Cells Dev., 16(1):7-23.
5. Stem cells: Resolving the self-renewal mystery. Sharma et al., 2008. Stem
cell Research (Under communication).
6. Effect of inhibitors on PI3K/RAS-MAPK/JNK pathway on stem cell
factor receptor (c-Kit) and its mutant on proliferation. Sharma et al., 2011.
(Under preparation).
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