AlanFung:LabNotes/Human MDA Halotyping/2009-7-20: Difference between revisions
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==Procedure== | ==Procedure== | ||
*Harvest the cells and transfer them to a 1.5ml tube | |||
*Centrifuge at 16000g for 10sec to pellet the cells | |||
*Remove the supernatant | |||
*Add 200ul pbs to wash the cells repeat centrifuge and remove pbs | |||
*Vortex vigorously to resuspend cells????? | |||
*Add 600 ul of nuclei lysis solution and pipet to lyse the cells (pipet until no visible cell clumps remain) | |||
*OPTIONAL add 3ul of rnase solution to the nuclear lysate and mix the sample by inverting the tube 5 times uncubate the mixture for 30m 37C allow the sample to cool to rt for 5m before proceeding | |||
*add 200ul of protein precipitation solution and vertex vugorously at high speed for 20sec | |||
*Chill sample on ice for 5m | |||
*centrifuge for 4n at 16000g | |||
*add 600ul rt isopropanol to a 1.5ml tube | |||
*transfer the supernatant containing the DNA to the 1.5ml tube | |||
*gently mix the solution by inversion until the white thread like strands of dna form a visible mass | |||
*centrifuge for 1m at 16000g at rt | |||
*decant the supertant | |||
*add 600ul of rt 70% ethanol and gently invert the tube several times to wash the dna | |||
*centrifuge for 1m at 16000g at rt | |||
*decant the supernatant | |||
*invert the tube on clean absorbent paper and air dry h pellet for 15m | |||
*add 100ul of dna rehydration solution | |||
*incubate at 65C for 1 hr periodialy mix the solution by gently tapping the tube | |||
*store dna at 4C | |||
='''PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)'''= | ='''PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)'''= |
Revision as of 14:53, 20 July 2009
Genomic DNA Extraction from GM18506 cells
Samples and Materials
- Wizard Genomic DNA purification kit
- 1.5mL microcentrifuge tubes
- 15 mL centrifuge tubes
- PBS
- 37C water bath
- Isopropanol
- 70% ethanol
- 65C water bath
Procedure
- Harvest the cells and transfer them to a 1.5ml tube
- Centrifuge at 16000g for 10sec to pellet the cells
- Remove the supernatant
- Add 200ul pbs to wash the cells repeat centrifuge and remove pbs
- Vortex vigorously to resuspend cells?????
- Add 600 ul of nuclei lysis solution and pipet to lyse the cells (pipet until no visible cell clumps remain)
- OPTIONAL add 3ul of rnase solution to the nuclear lysate and mix the sample by inverting the tube 5 times uncubate the mixture for 30m 37C allow the sample to cool to rt for 5m before proceeding
- add 200ul of protein precipitation solution and vertex vugorously at high speed for 20sec
- Chill sample on ice for 5m
- centrifuge for 4n at 16000g
- add 600ul rt isopropanol to a 1.5ml tube
- transfer the supernatant containing the DNA to the 1.5ml tube
- gently mix the solution by inversion until the white thread like strands of dna form a visible mass
- centrifuge for 1m at 16000g at rt
- decant the supertant
- add 600ul of rt 70% ethanol and gently invert the tube several times to wash the dna
- centrifuge for 1m at 16000g at rt
- decant the supernatant
- invert the tube on clean absorbent paper and air dry h pellet for 15m
- add 100ul of dna rehydration solution
- incubate at 65C for 1 hr periodialy mix the solution by gently tapping the tube
- store dna at 4C
PCR validation for 200pg gDNA limited MDA amplicons-061709 (P1-P12)
Procedures
- Experiment design
- Primers: Human genome specific primers set 1 (primer 1 to 12)
- Templates: 200pg MDA amplicons from 06/16/09 200pg MDA: L4N7, N6 in 1/50 dilution. Pos (100 pg/uL gDNA), NTC (RT-PCR grade H2O)
- Prepare diluted primers in PCR tubes (24 tubes)
For each primer needs 7+1 reaction 1rxn (7+1) rxns Primer mix (f+r 10 uM) 1.0 8.0 uL H2O 7.0 56.0 uL --------------------------------------------- 8.0 64.0 uL
- Prepare master mix in 1.5-mL microcentrifuge tube, wrapped with foil
1rxn (12+2) rxns x 7 Econo (2X) 10.0 140.0 Template 2.0 28.0 – (N6, 15C, 20C, 1-100dNTP, 1-1000dNTP, Pos, H2O) ----------------------------------- 12.0 168.0 168/14=12
- Mix well the master mix by vortexing. Transfer 12 uL Master mix into each of 12 reactions.
- Transfer 8 uL diluted primer into each reaction based on the arrangements
- Gently vortex and spin down the PCR reaction tubes. Place in Chromo4 machine immediately to perform realtime PCR reaction.
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 50 -> 72C 10min -> 15C hold
PCR test on the whole set of human genome primers
Background
- Testing if the human genome primers (f+r 10uM premixed) were degraded.
Procedures
- Primers: Human genome specific primers set 1 (primer 1 to 24)
- Templates: Pos (1 ng/uL gDNA)
1rxn Primer mix (f+r 10 uM) 1.0 uL H2O 7.0 uL Template 2.0 uL Econo (2X) 10.0 uL --------------------------------------------- 20.0 uL Amplified at 52C for 40 cycles
Gel electrophoresis
- 110mL (0.5X TBE) 2% agarose + 8 uL SYBR safe -> 2 middle gel with 26-well comb
- Loading: Sample 7uL + 6X loading buffer 1.5 uL (Order: P1 -> P24 left to right)
- Run at 135V for 20 min
File:ZhangLab 2 2009-07-16 14hr 23min pos-test - annotate.jpg
- All of the human genome primers are still working fine. 40 cycles amplification with 7uL sample loading amount will be used as the standard for following PCR tests