Primers
Put recently arrived primers in solution to 100uM using TE buffer (added and vortexed).
PCR
Ran PCRs for the following samples:
Product | Gene | Starting Plasmid | Forward Primer | Reverse Primer | Tm hi/lo (anneal) |
|---|---|---|---|---|---|
| CD79A | CD79A | pENTR CD79A (pDONR223) | iGEM KRB 003 F | iGEM KRB 004 R | 62/61 (61) |
| CD79A-TCS | CD79A | pENTR CD79A (pDONR223) | iGEM KRB 003 F | iGEM KRB 005 R | 62/61 (61) |
| mKate | mKate | pENTR_L1_mKate_L2 | iGEM KRB 006 F | iGEM KRB 007 R | 58/61 (58) |
Ran PCRs according to protocol on NEB website: https://www.neb.com/protocols/2012/09/06/protocol-phusion-high-fidelity-pcr-master-mix-with-hf-buffer-m0531
Used 20 uL reaction. For plasmids, diluted to concentration 1ng/uL in EB buffer and added 1uL of each dilution to a separate PCR mix. Diluted primers from 100uM stocks to 10uM with TE buffer.
PCR recipe:
| Quantity | Chemical |
| 1 uL | forward primer (10uM) |
| 1 uL | reverse primer (10uM) |
| 1 uL | template DNA (1 ng/uL) |
| 10 uL | Phusion High Fidelity PCR master mix |
| 7 uL | dH2O |
| 20 uL | TOTAL |
Settings for thermocycler (program: PHUSION):
| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 35 Cycles | 98°C 60°C 72°C | 10 seconds 30 seconds 60 seconds |
| Final Extension | 72°C | 5 minutes |
| Hold | 4°C |
Electrophoresis Gel
Prepared electrophoresis gel according to protocol: Gel Extraction (includes making gels)
Made 50mL of agarose/TAE/SYBR-Safe solution (enough for one gel).
Loaded lanes as follows:
Lane 1: Hyperladder 1kb (5uL)
Lane 2: CD79A + stop codon (724bp)
Lane 3: CD79A + TCS (718bp)
Lane 4: linker + mKate (768bp)
When loading wells with PCR products, used Parafilm to mix 1uL of 6X Orange dye with 5uL of sample and loaded 5uL of this mixture in a lane. Ran gel at 120V for 30 minutes.
