Daniel:Notebook/ComboLock/2017-3-8
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Padlock0601 Test (Started Wed March 1)
Bglii Digest (Sense)
From the results last week, I realized that the original primer for Bglii cutting (the DNA must be double stranded and the RCA product is single stranded) was in the wrong sense. This is because the RCA product will be the reverse complement of the circular product. So I'm repeating the Bglii digest from last Thursday using the correct primer.
- BglII Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 16.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make 17.2X qPCR master mix according to following recipe
- 361.2 uL nfH2O
- 430 uL SYBR Fast
- 17.2 uL 10 uM AmpF
- 17.2 uL 10 uM AmpR-Ind21
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout [[Image:|600px]]
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
Reagent | Single Rxn Vol | 16.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 2 | 32.4 |
10X Buffer 3.1 | 2 | 32.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 162 |
Total | 20 | 226.8 |