Objective
- Run gel with test PCR reactions with differing reagents
- Glycerol stock overnight growths: pMAL-pIII and pMXB10 in DH10B and ER2566
- Optional miniprep of DH10B growths
- Innoculate 2 x 500mL 2xYT media (+ 500μL 100mg/mL) with 10mL of 50mL overnight growths in ER2566 (grow, shaking at 37°C)
- Induce expression when OD @600nm reaches 0.6 (about 2hours)
- Grow overnight, shaking at 37°C
Bench work
- Made glycerol stocks of the overnight growths from last night
- pMAL-pIII in DH10B + Amp → tube #112
- pMXB10 in DH10B + Amp → tube #113
- pMAL-pIII in ER2566 + Amp → #114
- pMXB10 in ER2566 + Amp → #115
- Miniprepped DH10B samples of pMXB10 and pMAL-pIII (...concentrations not checked, run on gel)
- DNA gel (1% agarose):
- Lane 1: Ladder (10μL)
- Lane 2: ----
- Lane 3: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer A and Pfu Turbo polymerase 1 8/16
- Lane 4: ----
- Lane 5: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer A and Pfu Turbo polymerase 2 8/16
- Lane 6: ----
- Lane 7: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer B and Pfu Turbo polymerase 1 8/16
- Lane 8: ----
- Lane 9: 5μL test PCR pCMV-SPORT6+BSA with Pfu Turbo buffer B and Pfu Turbo polymerase 2 8/16
- Lane 10: ----
- Lane 11: pMXB10 miniprep from today, 8/17
- Lane 12: ----
- Lane 13: pMAL-pIII miniprep from today, 8/17
- Innoculated 2 x 500mL of 2xYT media (+ 500μL 100mg/mL ampicillin) with 10mL of the 50mL ER2556 growths from last night (pMAL-pIII and pMXB10)
- After a little under 2 hours, checked the OD @600nm and they were all overgrown by a good bit:
- pMXB10 #1 = 0.764
- pMXB10 #2 = 0.755
- pMAL-pIII #1 = 0.663
- pMAL-pIII #2 = 0.657
- Immediately induced expression with 500μL 1M IPTG in each flask
- Let grow, shaking overnight @37°C
- Made buffers for protein purification
- 1M TrisCl pH 8.0, 100mL
- 12.114g, used HCl to balance to pH 8.0; filtered through 0.45μm membrane
- 30mM TrisCl pH 8.0 with 20% w/v sucrose, 200mL
- 6mL of 1M TrisCl stock, 40g of sucrose, balanced pH with HCl; filtered through 0.45μm membrane
Results
Gel
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AU CHEM-570 Lab Prep
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