To check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.
Seed pET43.1 DH5α in LB + carbenicillin media 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&alpha combinations need to be stored beyond the lifetime of plate colonies.
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
• 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 (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer.
2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.
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 BamH I/Hind III
• 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.
Table 5
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
Results
The digestion has worked since the plasmids digested have moved faster.
NB: the gel suffered from overheating.
Aim:
Repeat Midiprep for pET43.1a(+). Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.
• Falcon of 50 ml
• Petri dish with DH5α transformed with pET43.1a(+)
• LB (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 µl of carbenicillin stock, to obtain 50 µg/µl.
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.
• 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 minutes (3500 RPM) and remove the supernatant.
2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 rpm during 7 minutes.
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, set point 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 600nm (OD600nm).
• Falcon of 50 ml
• LB autoclaved 500 ml
• LB (Luria Broth) autoclaved (500 ml)
&bull Precultures performed on the June 14, 2016
• Antibiotic: carbenicillin 100 mg/ml
• Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this link)
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 minutes onwards. We recorded OD600nm for this specific experiment after 3 hours.
Table 6
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
What we did on the lab:
Method:
1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 µl 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 synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer.
Method:
Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes.
Table 7
Name
Weight (mg)
Cfinal (ng/µl)
V(TE) (µl)
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. Spe I/ Xba I or BamH I/Hind III 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.
• 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:
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
3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min).
4. Load sample onto the column. Close the cap, spin for 1 minute then discard flow through.
5. Re insert column into collection tube. Add 200 µµl DNA wash buffer and spin for one minute. Discard flow-through.
6. Repeat step 5.
7. Transfer column to a clean 1.5 ml microfuge tube.
8. Add 6 µl of DNA Elution buffer to the center of the matric. Wait for 1 minute and spin for 1 minute to elute DNA.
Aim:
After digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2.
