Sam:LabNotes/Microbiome/2009-3-12 exp2
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Large scale MDA on diluted whole cells using - L4N6 primer plus Tre[edit]
Objective[edit]
- The L4N6 (locked nucleic acid primer) plus Tre (1.2 M) showed the best result of amplifying curves (on 3-10-09' exp) which can differenciate one single cell and background the most. The current experiment is designed to capture the diluted whole cell using the same set up.
Sample & Material[edit]
- Primer:L4N6 primer
- Cell: GM18506 cell line, harvested, washed by UV-treated PBS and coundted to 200,000 cell/mL (200 cell/uL)
- Dilute to 10 cell/uL, 5 cell/uL, 0.5 cell/uL using UV-treated PBS
Dilute factor original x 1/2 x 1/10 x 1/2 x 1/10 conc.(cell/uL) 200/uL 100/uL 10/uL 5/uL 0.5/uL Transfering(uL) 50 10 50 10 UVed-PBS(uL) 50 90 50 90
- Pos. control: human gDNA diluted to 3 ng/uL
- Measure the original (commercial human gDNA) using Nanodrop -> 309ng/uL
Dilute factor original x 1/100 x 1/10 x 1/10 x 1/10 conc. 309ng/uL 3ng/uL 300pg/uL 30pg/uL 3pg/uL Transfering(uL) 1 10 10 10 UVed-PBS(uL) 99 90 90 90
- Make fresh ALS buffer:(400mM KOH, 100mM DTT, 10mM EDTA)
- Prepare 5M KOH: weigh one KOH pallet in a 1.5mL-tube. Add nuclease-free H2O to make 5M KOH.
- Mix 20 ul 5M KOH, 5 ul 0.5M EDTA, 25 ul 1M DTT(Qiagen), 200 ul nuclease free H2O (Note: this is new formula)
- 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
0.093 g (KOH=56.11) = 0.001657 mole; 0.001657/5 x 10^6 =331.49 uL --- H2O needed
Reaction arrangement[edit]
- Left(A) to Right(H)
- Row1: 3ng, 300pg, 30pg, 3pg x 3, H2O x 2
- Row2: 0.5 cell x 8
- Row3: 0.5 cell x 8
- Row4: 0.5 cell x 6, 10 cell, 5 cell
- Total = 32 rxn -> Prepare (8+1)x4 =36 rxns for master mix
Procedures[edit]
- Thaw the reagents on ice and UV-treated H2O, 1XPBS and equipment for 10 min
- The the cool block out from frezor and leave in room tempterature for at least 10 min (To prevent the over-cold problem)
- Dilute the gDNA in UV-treated H2O into 3ng/uL, 300pg/uL, 30pg/uL, 3pg/uL
- Prepare ALS buffer and test the pH (use old NS buffer is ok)
- Transfer 250 uL(or enough amount) of ALS, NS, and dH2O into PCR tubes and UV-treated for 10 min (using cross linking machine)
- At the same time, dilute the cell with UV-treated PBS into 10 cell/uL, 5 cell/uL and 0.5 cell/uL
- Prepare the 2X SYBR (2uL of 50X SYBR + 48 uL H2O)
- Prepare MDA master mix (in a 0.6-mL tube)and leave on ice
1 rxn x 36 rxn ------------------------------------------------- H2O 6.2 uL 223.2 uL 1mM L4N6 primer 1.0 uL 36.0 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 ------------------------------------------------- 432.0 uL (432/36=12)
- Prepare cell lysates:
- Trasfer 1 uL ALS buffer into each of 32 PCR tubes on the cool-block
- Transfer 1 uL template (Pos, Neg, diluted whole cell) into each of 32 PCR tubes as prevous indication, mix with pipetting in each transferring.
- Incubate for 5 min on cool block
- Transfer 1 uL NS buffer into each of 32 PCR tubes, mix with pipetting.
- Transfer 5 uL of Tre(1.2M) into each of 32 PCR tubes, mix with pipetting.
- Transfer 12 uL MDA master mix to each of 32 PCR tubes. Mix with pipetting.
- Perfomr realtime PCR in Chromo4 with the new program
line 1. Incubate at 30C for 6 min line 2. Plate read line 3. Goto line 1 for 99 more times line 4. Incubate at 30C for 6 min line 5. Plate read line 6. Goto line 4 for 99 more times line 7. Incubate at 85C for 3 min line 8. Incubate at 4C forever
Note: Tre treatment will delay the reaction time, so we put 200 cycles in total. (The maximum setting is 99 cycles for the program)
- Stop the program (when the reaction reach the signal saturation) and pick up the samples
- Inactivate the samples at 85C for 3 min using thermocycler.
Results[edit]
File:Sam031209-L4N6 Tre.bmp Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction