Sam:LabNotes/Microbiome/2009-2-25/exp2: Difference between revisions
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>Sam Chiang (New page: ==Perform MDA on human cell lysates - test 2== *Samples: **Fresh lymphocytes, GM18507 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL **UV-treated (10 min) 1X PBS - 10 mL (prepared in 15-mL Corning...) |
>Sam Chiang |
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Dilution Factor Start 1/10 1/40 1/400 | Dilution Factor Start 1/10 1/40 1/400 | ||
---------------------------------------------------------- | ---------------------------------------------------------- | ||
Cell suspension 100 uL 100 uL 10 uL 10 uL | |||
PBS 900 uL 30 uL 90 uL | PBS 900 uL 30 uL 90 uL | ||
---------------------------------------------------------- | ---------------------------------------------------------- | ||
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**Positive controls: 6 No-template control: 2 | **Positive controls: 6 No-template control: 2 | ||
**Total reaction: 32 x 20ul | **Total reaction: 32 x 20ul | ||
===Set up MDA experiment=== | ===Set up MDA experiment=== |
Revision as of 17:09, 11 March 2009
Perform MDA on human cell lysates - test 2
- Samples:
- Fresh lymphocytes, GM18507 - 2x10^5 cell/mL (= 200 cell/uL), 1 mL
- UV-treated (10 min) 1X PBS - 10 mL (prepared in 15-mL Corning tube)
Lymphocytes pre-processing
- Take cultured lymphocytes suspension from one T25 flask
- Aliquot 100 uL suspension for cell counting using the cell counter (Beckman)
- Withdraw the desired cell amount (2 x 10^5 cells) and wash using UV-treated 1XPBS twice (Spin at 1000rpm for 3min)
- Resuspend in 1 mL UV-treated PBS
- Make dilution to 10 cell/uL using UV treated PBS:
Dilution Factor Start 1/10 1/40 1/400 ---------------------------------------------------------- Cell suspension 100 uL 100 uL 10 uL 10 uL PBS 900 uL 30 uL 90 uL ---------------------------------------------------------- Concentration 200/uL 20/uL 5/uL 0.5/uL Mix by repeat pipetting using p200 pipettor in each step To decrease the error in the last dilution (1/100 -> 1/1000), this step is performed in four replicates, and labeled #1~#4
- Realtime PCR program set up (Number of reactions)
- cell lysate: 0.5-cell x 24
- Positive controls: 6 No-template control: 2
- Total reaction: 32 x 20ul
Set up MDA experiment
- Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
- Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
- Mix 8 ul 5M KOH, 2 ul 0.5M EDTA, 10 ul 1M DTT, 80 ul nuclease free H2O;
- Mix 10ul ALS buffer with 10ul NS buffer, check pH with a pH paper, adjust ALS buffer with KOH or H2O if pH is not within 7-8
- Decontamination with UV-treatment
- Aliquot ALS buffer and NS buffer to two individual PCR tube, with 100 uL of each buffer.
- Treat the buffer with UV for 10 min.
- Prepare MDA master mix (in a 1.5-mL tube):
1 rxn x 36 rxn ------------------------------------------------- H2O 6.2 uL 223.2 uL 200 uM N6 primer 5.0 uL 180.0 uL 10x RepliPhi phi-29 buffer 2.0 uL 72.0 uL 25mM dNTP 0.8 uL 28.8 uL 2X SYBR Green I 1.0 uL 36.0 uL RepliPhi Phi-29 (100U/ul) 1.0 uL 36.0 uL ------------------------------------------------- 576.0 uL (576/36=16) Leave on ice for later use
- Prepare cell lysates:
- Transfer 1 uL diluted cell (0.5 cell/uL) to each of 32 tubes in four 8-well strips. Diluted cell #1 goes to strip #1, and so on for the rest (#2 -> #2 strip...).
- Prepare Pos. and Neg. control: 8 reactions
- Transfer 1 uL of 20 cell/uL sample (x1) , 5 cell/uL sample (x1)
- gDNA 1 ug/uL (x1), 100 pg/uL (x1), 10 pg/uL (x1), 1 pg/uL (x1)
- DNAse-free H2O (x 2)
- DNA denaturing and nutralization:
- Add 1.5 uL ALS buffer into 1 uL diluted cells and mix by repeat pipetting (~3 times), incubate at RT for 3 min or until all tubes are finished (Time it!)
- Add 1.5 uL NS buffer, mix by repeat pipetting. Place the tubes on cool rack.
- Add 16 uL MDA mix to each of the sample tube on cool block. Mix completely by repeat pipetting.
- Incubate at 30C for 10h on Opticon4, take one fluorescent read every 6 minutes.
Results
File:Sam022509-graph only2-with annotation.bmp Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction
Discussion
- Nine out of 24 reactions (with 0.5 cell/uL template) were pick up for regular PCR confirmation
- A3, A6, A7, B4, B7, C3, C4 and C8
- One Blank reaction (D8) failed due to insufficient Master mix. Need to increase the master mix volume for this design.
- The back ground amplification (Ct=26~28)still occur in Blank control, althoough the UV-treated H2O (from phi29 kit) was used as template.
- ALS and NS buffer were UV-treated. All of the phi29 reagents kit except dNTP and enzyme were UV-treated (10 min).
- It is possible the contamination is from the Master mix.