232. Measure the amount of DNA extracted from the miniprep
233. Harvest the culture with Midiprep
234. Growth of bacteria
235. Extraction of plasmid DNA
Aim: Measure the quantity of plasmid using a Nanodrop (Thermofisher) before sending for sequencing (inserts B1 and C2) Protocol: follow in this link What we did in the lab: Materials: • Nanodrop (Thermofisher) • Elution buffer from QIAGEN kit • Microbiology equipment (Follow this link) Method: Analyze absorbance at 260nm Clean the Nanodrop with water Make the blank with 1ul of elution buffer Put 1ul of your sample on the Nanodrop Make the measure and clean the Nanodrop between each measure Results:
| Absorbance at 260nm | A260 | A280 | A260/280 | Concentration (ng/µL ) |
|---|---|---|---|---|
A1(1) |
0.202 | 0.124 | 1.62 | 10.1 |
A1(2) |
0.259 | 0.169 | 1.53 | 13.0 |
A2(1) |
0.179 | 0.105 | 1.70 | 8.9 |
A2(2) |
0.179 | 0.103 | 1.73 | 8.9 |
A2(3) |
0.098 | 0.052 | 1.86 | 4.9 |
A2(4) |
0.152 | 0.099 | 1.53 | 7.6 |
Aim: To start a culture for Miniprep of insert C2 in pET43.1a in BL21(DE3) from 22/08 and A1/C2 in pET43.1a in TOP1O. In order to obtain a large amount of plasmid, we need to grow the bacteria overnight. Protocol: follow in this link What we did in the lab: Materials: • Microbiology equipement • 50mL erlenmeyers • Carbenicillin 50 mg/ml • LB medium Method: One colony is picked from the plates and shaken in 25.0 mL of LB supplemented with Carbenicillin at 50 µg/ml. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight.
Aim: Produce our protein in BL21(DE3) competent cells, the production is induced with IPTG once it reaches an optical density of 0.7. Protocol: follow in this link What we did in the lab: Materials: • 2 2L erlenmeyers • LB (lysogeny Luria broth) • IPTG (0.1M) • Precultures in 25ml erlenmeyers • UV spectrophotometer (Ultrospec 3100) • Shaking incubator (INFORS HT) • Centrifuge • Buffer A (50mM Tris, 150mM of NaCl) Method: Put 1L of LB in each erlenmeyer and make them warm with the shaking incubator at 37°C and 150RPM Once warmed, add 12.5ml of preculture in each erlenmeyer Let grow in the shaking incubator. Measure the absorbance with the UV spectrophotometer every 30min
| Time | C2(1) | C2(2) |
|---|---|---|
9:36AM |
0.024 | 0.023 |
10:06AM |
0.049 | 0.046 |
11:02AM |
0.175 | 0.165 |
11:36AM |
0.395 | 0.402 |
12:05AM |
0.568 | 0.540 |
12:27AM |
0.658 | 0.638 |
12:38AM |
0.694 | 0.680 |
14:31PM |
0.872 | 0.859 |
Aim: To perform a Midiprep to isolate plasmid DNA of pET43.1a with the inserts A1/C2 from cultures made on the 1/09. The amplification method to increase the amount of plasmid is called Midiprep. Protocol: follow in this link What we did in the lab: Materials: • 50 ml Falcon tube • Shaking incubator (INFORS HT) • Swing bucket centrifuge (JOUAN GR41) • QIAGEN Miniprep kit • Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link) Method: The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 8 minutes. We will follow most of the protocol of QIAGEN Miniprep 2016 except for a few modifications, which we describe, therefore, below. Follow QIAGEN kit steps. Final volume: 100 µL
