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interactome:experiments:20191122-2 [2019/11/22 17:48] dan [Table] |
interactome:experiments:20191122-2 [2019/11/22 17:58] (current) dan |
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| ## RNA extraction from HEK293T cells | ## RNA extraction from HEK293T cells | ||
| - | {{tag>protocol RNA kit interactome PersonalInteractome}} | + | {{tag>interactome PersonalInteractome}} |
| #### Material | #### Material | ||
| 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 | ||
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| ^ Total ^ 10 ^ 100 | | | ^ Total ^ 10 ^ 100 | | | ||
| + | *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 197: | 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 per 1 reaction | ^ Note ^ | ||
| - | | | | | | | ||
| - | | 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 | ||
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| ^ 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 222: | 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 | | | ||
| \\ | \\ | ||
| \\ | \\ | ||
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| \\ | \\ | ||
| ==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== | + | |