Daniel:Notebook/ComboLock/2017-3-6
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Padlock0601 Test (Started Wed March 1)[edit]
After reading some papers I'm going to give it one more go to see if I can't amplify the DNA from last week better. I'll use the method of largest amplification I know: most time using random hexamers as primers.
RCA Repeat (Maximum Power!)[edit]
- Maximal Rolling Circle Amplification
- Create master mix according to following table
- Add 16 uL master mix to each tube
- Add 4 uL template according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 15 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Hexamer (1 mM) | 0.5 | 8.1 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 7.1 | 115.02 |
Total | 20 | 243 |
Continued tomorrow