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=Large scale MDA on diluted whole cells using - L4N6 primer plus Tre'''= ==Objective== *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== *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== *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== *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== [[Image:sam031209-L4N6_Tre.bmp|700px]] '''Note: Bewteen 5 cell/uL and Blank are amplification curves of 0.5 cell/uL reaction'''
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