On the June 6, 2016, we have done a transformation of pET43.1a(+) into DH5alpha and we dose it to know it concentration.
What we did in the lab:
Method:
The culture for plasmid amplification was carried out in a large volume then aliquoted in several 1.5 ml Eppendorfs for centrifugation, since our Falcon size rotors do not go up high in G force.
We have nine 1 ml Eppendorf with 50L of pET43.1 in each tubes so we assemble all tubes in one.
1. Centrifuge the first Eppendorf and pour it into the second Eppendorf.
2. Do the same protocol until the last Eppendorf.
3. Use a plastic uvette (path length 1cm) to measure the DNA concentration.
4. Put in 1ml of TE1X with P1000, then take off 15L with P20 and add 15L of DNA with P20.
5. Analysis in spectrophotometer, Eppendorf biophotometer plus at 260 nm
Results:
C1=87 ng/l
C2 = 103.3 ng/l
Due to these bad results, we decided to use another spectrophotometer “Eppendorf Biophotometer”:
C1=0,2 ng/l
C2= 0,8 ng/l
Again, our results were not correct so we remeasure with utraspec 3100 pro from Amersham Bioscience.
Table 10
Sample 1
Sample 2
Sample 3
A260
0.043
0.59
0.037
A280
0.026
0.01
0.016
A260 /A280
1.7
5.9
2.31
Cdilué(ng/µl)
2.15
1.95
1.85
Cfinal(ng/µl)
143.33
130
123.33
Cmedium(ng/µl)
133.22
133.22
133.22
We keep these results for the end of the experiment.
Then, we resuspend the insert by adding buffer TE1X in each tube (refer to the June 15, 2016 for the volumes).
Results:
Colonies on the petri dish were counted. We saw bacterial colonies so the stab culture was successful.
Refer to June 16, 2016, for protocol (dephosphorylation, ligation with C1 and C2 and transformation).
We spread the mix in petri dish with CB50 and CM34 to see if the transformation was successful.
Results:
We analyse the transformation done yesterday. We don’t saw bacteria colonies on the dish, so transformations were not successful. To understand why bacterial colonies don’t grow up on petri dish, plasmid DNA is assayed.
Aim:
To link pET43.1a(+) with C1 and C2 we assayed before plasmid DNA but perhaps our ligation doesn’t work. We want to know the concentration of our samples.
1. We need more buffer solution so we dilute TE 10X by ten to have TE.
In a 250 ml burette, we had 25ml of TE and we complete with water until 250 ml. We aliquot the 250 ml in fixe 50 ml bottle.
2. We made a 15/1000 dilutions with our samples (15 l of DNA and 985 l of water)
3. In a plastic uvette, we pour 1 ml of buffer TE, then we take back 15 l and add of DNA
4. We analyse at 260 nm
Table 11
λ= 260 nm
Sample 1
Sample 2
A260
A280
A260 /A280
Cdilué(ng/µl)
1.25
1
Cfinal(ng/µl)
250
200
Cmedium(ng/µl)
225
225
225
Aim:
Our transformation do not work so we made a new digestion with BamHI and Hind III. This step will let us to do the transformation..
1. Add all reagent in a 1 ml Eppendorf (1 for C1, 1 for C2 and the last one for pET43.1)
2. Let digest during 1h at 37°C then incubate 5 min at 65°C
For the reagent volumes, refer to the table.
Table 12
pET43.1= 130 ng/L
BamHI= 20 000 U/ml
C1/C2= 10 ng/L
Hind III= 20 000 U/ml
Aim:
This step is needed to purify our DNA. Indeed, Multiclonal site (parts digested by enzymes but useless) cannot be removed with a PCR clean up because their molecular weight is too low.
1. Make an 0,7% agarose gel (refer to June 13, 2016)
2. Fill the electrophoresis chamber with TAEO,5X buffer
3. Follow the deposit table:
Voltmeter: Voltmètre PowerPac HC Biorad
V = 50V
Amp(T=0) = 0.01 A
Table 13
pET43.1a(+)
C1
C2
pET43.a(+)(ng/µl)
23 µl
C1 or C2 10 ng/µl (400 ng)
40 µl
40 µl
BamHI 20 000 U/ml (10 U)
0.5 µl
0.5 µl
0.5 µl
HindIII 20 000 U/ml (20 U)
1 µl
1 µl
1 V
Tampon
5 µl
5 µl
5 µl
TOTAL
50 µl
50 µl
50 µl
Table 14
Lines
L1
L2
L3
L4
L5
L6
L7
L8-L9
L10
L11
Name
Molecular weight
pET43.a(+) uncut
pET43.1a(+) B
pET43.1a(+) H
pET43.1a(+) B/H
C1
C2
V(DNA) (µl)
15
15
15
15
15
15
15
V(H20) (µl)
0
0
0
0
0
0
0
V(Load buffer) (µl)
3
3
3
3
3
3
3
Results:
Figure 1
Aim:
We want to take back the plasmid and the inserts digested.
The electrophoresis has set up the waste and the parts needed
1. Cut agarose gel to have only parts of plasmid that we need and put it on an 1ml Eppendorf
Table 15
Empty eppendorf (g)
Eppendorf + gel (g)
Final weight (mg)
m (pET43.1a(+)
0.99
1.12
127.2
m(C1)
0.99
1.07
76.5
m(C2)
0.99
1.09
106.1
2. We use NEB extraction kit: De Macherey-Nagel_nucleospin-Gel and PCR Clean-up
For NT1 volumes: we need 200L of NT1 for 100 mg DNA
For pET43.1 volume = 254 l of NT1 buffer with p1000
For C1 volumes= 153 l of NT1 buffer with p1000
For C2 volumes= 212 l of NT1 buffer with p1000
Aim:
After the digestion we dephosphorylate It to prevent that it binds without the insert.
refer to June 6, 2016 but with these quantities:
- L of bacteria
- L of DNA
- L of SOC
Spread each sample on a petri dish made of LB and CB50 (carbenicillin 50g/ml)
Aim:
We want to assay DNA of C1 and C2 but we do not have enough insert so we tried different dilutions.
What we did in the lab:
Method:
1. First, we use 1 μl of C1 and 49 μl of water.
2. Make the blank with H20.
3. Use the spectrophotometer Utrospec 3100 pro_amersham Bioscience at 260 nm
4. Results were not convincing so we decided to use a Nanodrop
