Differences
This shows you the differences between two versions of the page.
| Both sides previous revision Previous revision Next revision | Previous revision | ||
|
interactome:experiments:20191122-2 [2019/11/22 17:42] dan [Table] |
interactome:experiments:20191122-2 [2019/11/22 17:58] (current) dan |
||
|---|---|---|---|
| Line 1: | Line 1: | ||
| ## RNA extraction from HEK293T cells | ## RNA extraction from HEK293T cells | ||
| - | {{tag>protocol RNA kit interactome PersonalInteractome}} | + | {{tag>interactome PersonalInteractome}} |
| #### Material | #### Material | ||
| Line 23: | Line 23: | ||
| * Freshly prepared DNaseI solution included in the kit | * Freshly prepared DNaseI solution included in the kit | ||
| - | ^ Check ^ Reagent ^ ^ 1 reaction (µL) ^ _ _ _ _ reaction (µL) ^ Memo ^ | + | ^ Check ^ Reagent ^ ^ 1 reaction (µL) ^ 2 reaction (µL) ^ Memo ^ |
| - | | | ddH2O (RNAse free) | | | | | | + | | | ddH2O (RNAse free) | | 87.5 | 175 | | |
| - | | | 10x DNase Buffer | | 10 | | | | + | | | 10x DNase Buffer | | 10 | 20 | | |
| - | | | DNaseI | | 30 units | | | | + | | | DNaseI | | 2.5 | 5 | 30 units. 12 units/µL | |
| - | ^ ^ Total volume (µL) ^ ^ 100 ^ ^ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ ^ | + | ^ ^ Total volume (µL) ^ ^ 100 ^ 200 ^ ^ |
| \\ | \\ | ||
| Line 151: | Line 151: | ||
| ## 1st strand cDNA synthesis of RNA samples extracted on 20191122 | ## 1st strand cDNA synthesis of RNA samples extracted on 20191122 | ||
| - | {{tag>protocol RNA cDNA kit interactome PersonalInteractome}} | ||
| #### Material | #### Material | ||
| Line 180: | Line 179: | ||
| - | ^ Reagent ^ Volume ^ Note | | | + | ^ Reagent ^ Volume for 1 reaction (µL) ^ Volume for 10 reactions (µL) ^ Note ^ |
| - | | Oligo dT Primer（50 μM） | 1 µL | | | | + | | Oligo dT Primer（50 μM） | 1 | 10 | | |
| - | | or Random 6 mers（50 μM） | or 1 µL（0.4～2 µL） | For genes <2kbp, use1～2 μl. When >2 kb, 0.4～1 μl is recommended. | | | + | | dNTP Mixture（10 mM each） | 1 | 10 | | |
| - | | or Gene specific primer (1 µM) | or 1 µL | Final concentration at 0.1 μM. | | | + | | RNA Template | ~8 | 50 | Total RNA：<5 μg, polyA＋ RNA：<1 μg | |
| - | | === | | | | | + | | RNase Free dH2O | up to 10 | 30 | | |
| - | | dNTP Mixture（10 mM each） | 1 µL | | | | + | ^ Total ^ 10 ^ 100 | | |
| - | | RNA Template | ~8 µL | Total RNA：<5 μg, polyA＋ RNA：<1 μg || | + | |
| - | | RNase Free dH2O | up to 10 µL | | | | + | |
| + | *Aliquot to 10 x 10 µL | ||
| *Incubate at 65˚C for 5 mins | *Incubate at 65˚C for 5 mins | ||
| *Transfer tube to ice immediately | *Transfer tube to ice immediately | ||
| Line 199: | Line 196: | ||
| ==2 Reverse transcription == | ==2 Reverse transcription == | ||
| - | *Add the following reaction mix, and make the total volume 20 µL. | + | *Prepare master mix for reaction; |
| + | ^ Reagent ^ Volume per 1 reaction (µL) ^ Volume per 5 reaction (µL) ^ Note ^ | ||
| + | | 5×PrimeScript II Buffer | 4 | 20 | | | ||
| + | | RNase Inhibitor（40 U/μl） | 0.5 | 2 | 20U per reaction | | ||
| + | | PrimeScript II RTase（200 U/μl） | 1 | 5 | 200U per reaction | | ||
| + | ^ RNase Free dH2O ^ 4.5 | 22.5 ^ ^ | ||
| - | ^ Reagent ^ Volume ^ Note ^ | ||
| - | | Reaction mix from previous step | 10 μL | | | ||
| - | | 5×PrimeScript II Buffer | 4 μL | | | ||
| - | | RNase Inhibitor（40 U/μl） | 0.5 μL（20 U） | | | ||
| - | | PrimeScript II RTase（200 U/μl） | 1 μL（200 U） | | | ||
| - | ^ RNase Free dH2O ^ up to 20 μL ^ ^ | ||
| + | *Add 10 µL of the master mix to each tube to make the total volume 20 µL. | ||
| *Mix gently | *Mix gently | ||
| Line 215: | Line 212: | ||
| ^ Temprature ^ Time ^ Note ^ | ^ Temprature ^ Time ^ Note ^ | ||
| - | | 30˚C | 10 min | Only when using random 6mer primers | | + | | 45˚C | 60 min | | |
| - | | 45˚C | 30-60 min | | | + | |
| | 75˚C | 15 min | Deactivation. Paramater for long transcrpits just in case. | | | 75˚C | 15 min | Deactivation. Paramater for long transcrpits just in case. | | ||
| | 4˚C | Forever | (Immediately put on ice.) | | | 4˚C | Forever | (Immediately put on ice.) | | ||
| Line 224: | Line 220: | ||
| *The cDNA product should be stored at -20°C. | *The cDNA product should be stored at -20°C. | ||
| *For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume. | *For downsteam PCR amplification, the volume of cDNA product should not exceed 1/10 of the PCR reaction volume. | ||
| + | |||
| + | |||
| + | ^ Sample No. ^ Date prepared ^ Number of freeze ^ Number of thaw ^ "Done" if used ^ | ||
| + | | 1 | 20191122 | 1 | 0 | | | ||
| + | | 2 | 20191122 | 1 | 0 | | | ||
| + | | 3 | 20191122 | 1 | 0 | | | ||
| + | | 4 | 20191122 | 1 | 0 | | | ||
| + | | 5 | 20191122 | 1 | 0 | | | ||
| + | | 6 | 20191122 | 1 | 0 | | | ||
| + | | 7 | 20191122 | 1 | 0 | | | ||
| + | | 8 | 20191122 | 1 | 0 | | | ||
| + | | 9 | 20191122 | 1 | 0 | | | ||
| + | | 10 | 20191122 | 1 | 0 | | | ||
| \\ | \\ | ||
| \\ | \\ | ||
| Line 231: | Line 240: | ||
| \\ | \\ | ||
| ==Protocol last updated== | ==Protocol last updated== | ||
| - | --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/11/21 15:16// | + | --- //[[daney@sfc.keio.ac.jp|Dan Yamamoto-Evans]] 2019/11/22 17:55// |
| - | \\ | + | |
| - | \\ | + | |
| - | ==Protocol used== | + | |