AlanFung:LabNotes/Human MDA Halotyping/2009-7-20
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Genomic DNA Extraction from GM18506 cells[edit]
Samples and Materials[edit]
- 3 x 10^6 cells
- Wizard Genomic DNA purification kit
- 1.5mL microcentrifuge tubes
- 15 mL centrifuge tubes
- PBS
- 37C water bath
- Isopropanol
- 70% ethanol
- 65C water bath
Procedure[edit]
- 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)
- 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 the 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
- nanodrop
PCR validation for gDNA[edit]
sample and material[edit]
- human genome specific primers
- templates
- Jurkat gdna
- h2o
- ntc h2o
- econotaq
Procedures[edit]
' | GM18506 | Pos. | Neg. |
primer mix (f+r 10uM) | 1 | 1 | 1 |
h2o | 7 | 7 | 9 |
econotaq 2x | 10 | 10 | 10 |
Gm template 1ng/ul | 2 | 0 | 0 |
Jurkat template | 0 | 2 | 0 |
- 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 pcr machine immediately to PCR reaction.
- Program: 94C 2min-> (94C 40sec-> 52C 40sec -> 72C 60 sec) x 30 -> 72C 10min -> 15C hold
PCR test on the whole set of human genome primers[edit]
Background[edit]
- Testing if the human genome primers (f+r 10uM premixed) were degraded.
Procedures[edit]
- 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[edit]
- 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