5. Analysis and storage of the transformation done on June 6, 2016
6. Electrophoresis of pET43.1 and pSB1C3
7. Preculture of pET43.1a(+) DH5α to make stab culture
8. Make a culture of DH5α pET43.1 to monitor the growth curve
9. Make a growth curve of bacteria culture
10. Stab culture
11. Resuspension of C1 and C2 and all inserts synthesized by iDT
12. Extraction of pET43.1a(+) and pSB1C3 DNA from agarose gel
13. Digestion of inserts (C1 and C2)
14. Dephosphorylation
15. Ligation of C1 and C2 with pET43.1a(+)
Aim: Check the transformation and the digestion of the plasmids by electrophoresis on agarose gel. Seed pET43.1 DH5α in LB + carbenicillin to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5 combinations need to be stored beyond the lifetime of plate colonies. Protocol: follow in this link What we did in the lab:Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 µg/ml (LB + CB50) or with chloramphenicol 34 µg/ml (LB + CM34) were counted for the presence of individual distinct colonies. LB + CB50
| Box 1 | Box 2 | Box 3 | |
|---|---|---|---|
pET43.1a(+) DH5α |
0 | 2 | 23 |
| pET43.1a(+) DH5α (different batch) |
Uncountable (Overgrown) | 2 | |
pET43.1a(+) DH5α (different batch) |
Uncountable (Overgrown) | 15 |
| Box 1 | Box 2 | |
|---|---|---|
pSB1C3 DH5α |
1 | 78 |
| pSB1C3 DH5α (different batch) |
38 | Uncountable (Overgrown) |
What we did in the lab:
Materials:
• 1X TAE buffer
• Agarose
• Microwave oven /
• Distilled water
• Ethidium Bromide
• Gel caster, and power supply
• Precision balance
Method:
1. Buffer solution: we have 1.0X stock TAE solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer
2. Agarose gel: We prepared 0.7% w/v agarose gel.
For agarose gel electrophoresis experiment:
Samples:
• pSB1C3 plasmid (obtained with Midiprep on June 8, 2016)
• pSB1C3 plasmid (digested by SpeI/XbaI)
• pET43.1a plasmid (obtained with Midiprep on June 8, 2016)< /br>
• pET43.1a plasmid digested by BamHI/HindIII
• gel 0.7% agarose
• TAE 0.5X buffer
• Electrophoresis power supply
• DNA ladder (Thermofisher Gene ruler 1kb))
Method:
1. After filling the electrophoresis chamber with TAE 0.5X buffer, samples were loaded in 1X gel loading buffer in the wells, according to the following lane pattern.
| Lanes | L1 | L2 | L3 | L4-7 | L9-8 | L10-13 | L14 | L15 | |
|---|---|---|---|---|---|---|---|---|---|
Sample |
Molecular weight | pET43.1a(+) uncut | pET43.1a(+) B-H | pBS1C3 S-X | pBS1C3 uncut | ||||
| DNA (µl) | 5 | 2 | 2 | 4 | 4 | ||||
| H20 | 0 | 8 | 8 | 6 | 6 | ||||
| 6XLoadingbuffer | 0 | 2 | 2 | 2 | 2 |
Aim:Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.
Protocol: follow in this link
What we did in the lab:
Materials:
• Falcon of 50 ml
• Petri dish with DH5α transformed with pET43.1a(+)
• L.B (Luria Broth) medium autoclaved (5 ml)
• Shaking incubator (INFORS HT)
• Antibiotic: Carbenicillin 100 mg/ml
• Pipetman P10 and 5 ml graduated pipet
• Bunsen burner
• Cones for P10
• Propipet
• Rack for 50 ml tubes
Method:
1. Pick one colony and put it into 5 ml of LB medium + 2,5 of carbenicillin, to obtained 50
2. Grow overnight at 37°C in a shaking incubator at 150 rpm.
Aim: Use the preculture of June 13, 2016 to start the new culture.
Protocol: follow in this link
What we did in the lab:
Materials:
• Falcon of 50 ml
• LB autoclaved 500 ml
• 3 precultures performed on June 13, 2016 (redundant cultures in case colonies don’t grow)
• carbenicillin 100 mg/ml
• swing bucket centrifuge (JOUAN GR4i)
Method:
1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 min (3500 RPM) and remove the supernatant.
2. Add 5ml of fresh L.B, resuspend the pellet and centrifuge again at 3500 RPM during 7 min.
3. Repeat one more time step 2
4. Resuspend the pellet into 5 mL of fresh LB+ 2.5 µL of carbenicillin 100 mg/ml
5. Grow in the shaking incubator (T= 37 °C / 130 RPM) Overnight
We overshot the 0.7 OD, setpoint therefore we continued overnight.
Aim: to make a stab culture we need to capture the bacteria growing at the exponential phase In order to do that we need to take optical density at 600 nm (OD600nm).
Protocol: follow in this link
What we did in the lab:
Materials:
• Falcon of 50ml
• LB autoclaved 500 ml
• L.B (Luria Broth) autoclaved (500 ml)
&bull Precultures performed on the June 14, 2016
• Antibiotic: carbenicillin 100 mg/ml
• Microbiology equipment
Method: Before starting the culture we need to have molten LB-agar so we need to place the solid LB agar on a hot plate with stirring.
1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600nm every hour for the first three hours, then every 20 min onwards. We recorded OD600nm for this specific experiment after 3 hours.
| Time | Absorbance A’ | Absorbance A’’ | |
|---|---|---|---|
12h37 |
0.013 | 0.015 | |
| 13h07 |
0.023 | 0.027 | |
| 13h37 |
0.063 | 0.059 | |
| 14h07 |
0.110 | 0.105 | |
| 14h27 |
0.172 | 0.170 | |
| 14h47 |
0.265 | 0.256 | |
| 15h07 |
0.384 | 0.377 | |
| 15h27 |
0.491 | 0.482 | |
| 15h47 |
0.567 | 0.557 | |
| 16h06 |
0.697 | 0.608 | |
| 16h29 |
0.816 | 0.755 | |
| 17h03 |
0.954 | 0.909 | |
| 17h20 |
0.954 | 0.909 | |
| 17h40 |
1.061 | 1.029 | 18h00 |
1.092 | 1.082 | 18h00 |
1.122 | 1.132 |
Protocol: follow on this link What we did on the lab: Method: 1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 l of carbenicillin 50 mg/ml added, swirl to mix 2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total) 3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times 4. Grow overnight at 37 °C and next day place it at 4°C
Aim: we received our gene block synthetized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. Add TE buffer in each tubes and refer to the next table for volumes
| Name | Weight (mg) | Cfinal (ng/µ ;l) | V(TE) (µ ;l) |
|---|---|---|---|
A1 |
500 | 10 | 50 |
A2 |
500 | 10 | 50 |
C1 |
1000 | 10 | 100 |
C2 |
1000 | 10 | 100 | D1 |
500 | 10 | 50 | D2 |
500 | 10 | 50 | E1 |
500 | 10 | 50 | E2 |
500 | 10 | 50 | B2 |
1000 | 10 | 100 |
Aim: We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. SpeI/ XbaI or BamHI/HindIII respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN. Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2 Protocol: follow in this link What we did in the lab: Material: • We use the NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G) Method: 1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. 2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: .
| EmptyEppendorf (g) | Eppendorf+ gelf (g) | Final weight (mg) | ||
|---|---|---|---|---|
pET43.1a(+) |
m1 | 0.9 | 1.3 | 329.6 |
pET43.1a(+) |
m2 | 0.9 | 1.3 | 265.3 |
pSB1C3 |
m1 | 0.9 | 1.2 | 251.1 |
pSB1C3 |
m2 | 1.0 | 1.2 | 185.6 |
Aim: after digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2. Protocol: follow in this link What we did in the lab: Material: Method: Add all reagents in 1 ml Eppendorf - Digest during 2 h at 37 °C - For the reagent volumes, refer to the Table 9. Total volume = 50 µl
| C1 | C2 | ||
|---|---|---|---|
DNA (µl) |
20 | 20 | |
BamHI (µl) |
1 | 1 | |
HindIII (µl) |
2 | 2 | |
H20 (µl) |
22 | 22 | |
CutSmart(µl) |
5 | 5 |
