FastLine cell cdna Kit

Transcription

1 1. FastLine cell cdna Kit For high-speed preparation of first-strand cdna directly from cultured cells without RNA purification

2 RT FastLine cell cdna Kit Cat. no. KR105 Kit Contents Contents Buffer FCW Buffer FCP gdna Wipeout Buffer (7x) F-Quant Reverse Transcriptase QuantiScript RT Buffer (5x) RT Primer Mix RNase-Free ddh 2 O KR rxn 25 μl 10 μl 100 μl 50 μl 200 μl 50 μl 1 ml Handbook 1 Storage Buffer FCW and Buffer FCP should be stored at room temperature (15-25 C). All other components of the FastLine cell cdna Kit should be stored immediately upon receipt at -20 C in a constanttemperature freezer. Introduction The FastLine cell cdna Kit provides a high-speed procedure for generating first-strand cdna directly from cultured cells. No RNA purification or RNase H digestion steps are necessary, minimizing pipetting tasks. The FastLine cell cdna Kit supplies wash and lysis buffers for preparing lysates and stabilizing RNA; gdna Wipeout Buffer for eliminating genomic DNA contamination, and all reaction components for fast and efficient cdna synthesis. Important Notes 1. This product is developed for use with adherent cultured cells, FastLine cell cdna Kit Handbook 1

3 suspension cells and frozen stored cell. When using a 24-well plate, 4x10 4 cell typically need to be seeded per well. In general, other cell numbers are depending on cell type and culture conditions, different plate needs different quantity of cell, according to table 1. Table 1. Cell numbers needed in different plates Plate Cells seeded Buffer FCW / Buffer FCP / in plate / well well well 384-well plate μl 12.5 μl 96-well plate μl 50 μl 48-well plate μl 100 μl 24-well plate μl 200 μl 12-well plate μl 400 μl 6-well plate μl 1000 μl 2. Set up reverse transcription reactions on ice for optimal results. 3. After reverse transcription, the reaction must be inactivated by incubation for 3 min at 95 C to inactivate F-Quant Reverse Transcriptase. 4. For optimal results, 10 pg -1 μg RNA is recommended. With >1 μg RNA, scale up the reaction linearly to the appropriate volume. Protocol Protocol (a) is for adherent cells; protocol (b) is for suspension cells; protocol (c) is for frozen stored cells 1. (a) Adherent cells: Seed an appropriate number of fresh cells (eg. cells seeded in 24-well plate, with 4x10 4 cell per well). FastLine cell cdna Kit Handbook 2

4 (b) Suspension cells: Pipet an appropriate number of suspension cells in tub, centrifuge at 250 g for 5 min. (c) Frozen stored cells: Pipet an appropriate number of frozen stored cell in tub, take step 3 directly following protocol for Suspension cells. 2. (a) Aspirate the cell-culture medium using a pipet, and discard. (b) Discard the cell-culture medium after centrifugation. 3. (a) Add 500 μl Buffer FCW per well. (b) Add 500 μl Buffer FCW per tub. Note: Do not incubate/wash the cells with Buffer FCW for long periods of time. 4. (a) Aspirate Buffer FCW using a pipet, and discard. (b) Centrifuge at 250 g for 5 min, discard Buffer FCW. 5. (a) Add 200 μl Buffer FCP per well. Incubate for 5-10 min at room temperature (15-25 C). (b) Add 200 μl Buffer FCP per tube. Incubate for 5-10 min at room temperature (15-25 C). 6. Transfer the FastLine lysates into appropriately sized tubes. Proceed immediately to step 7. Note: If a pause in the procedure is required, store the tubes containing the lysates at -80 C. 7. Prepare the following solutions at room temperature (15-25 C): FastLine lysates, RNase-Free ddh 2 O, gdna Wipeout Buffer (7x), Quant Reverse Transcriptase, QuantiScript RT Bufer (5x) and RT Prime Mix. Note: Put Quant Reverse Transcriptase, QuantiScript RT Buffer (5x) and RT Primer Mix on ice after thawed. 8. Prepare the genomic DNA elimination reaction according to Table 2. Table 2. gdna Elimination Reaction Components FastLine cell cdna Kit Handbook 3

5 Contents Volume Final Concentration gdna Wipeout Buffer (7 ) 2 μl 1 FastLine lysate RNase-Free ddh 2 O Total Volume 1-4 μl Up to 14 μl 14 μl 9. Incubate for 5 min at 45 C. Then place immediately on ice. 10. Prepare the reverse-transcription master mix on ice according to Table 3. Table 3. Reverse-Transcription Reaction Components Contents Volume Final Concentration Quant Reverse Transcriptase 1 μl Quantiscript RT Bufer, 5x 4 μl 1x RT Primer Mix FastLine lysate 1 μl 14 μl Total Volume 20 μl 11. Incubate for 30 min at 42 C. 12. Incubate for 3 min at 95 C. 13. cdna can be used in the following real-time PCR. FastLine cell cdna Kit Handbook 4

6 Ordering Information RNA Isolation Product Size Cat. no. RNAprep Pure Cell/Bacteria Purification Kit 50 preps DP430 RNAprep Pure Tissue Purification Kit 50 preps DP431 RNAprep Pure Plant Purification Kit 50 preps DP432 Real-Time PCR Product Size Cat. no. SuperReal PreMix Plus (SYBR Green) 20 μl 125 rxn 20 μl 500 rxn FP FP PCR Product Size Cat. no. 2 Taq PCR MasterMix (with loading dye) Fast HiFidelity PCR Kit 1 ml 5 1 ml 50 μl 50 rxn 50 μl 200 rxn KT KT KP KP FastLine cell cdna Kit Handbook 5