We want to extract the DNA we tried to transform on June 22, 2016.
The aim is to obtain the construct of C1 in pET43.1 for expression in E. coli. This to serve both the verification of the presence of the pET43.1-C1 plasmid in the cells but also to make a stock of it for future cloning and expression experiments.
•pET43.1-C1 obtained by the midiprep kit
•plastic uvette (Eppendorf)
•spectrophotometer
•TE (Tris 10 mM pH 8.0, EDTA 1 mM) buffer
Method:
1. Use a 1/50 dilution of pET43.1-C1 plasmid in TE, volume =500 µl.
2. We use the spectrophotometer ultrospec 3100 pro- Amersham Bioscience and we do the blank with TE.
Table 18 : absorbance of DNA measured at 260nm, and using ds-DNA (double stranded DNA) absorbance of 50 µg/ml for an optical density of 1 (A260nm=1)
λ= 260nm
pET43.1a(+) C1
A 260 nm
0.065
A 280 nm
0.57
A 260 nm/
A280 nm
1.14
Cfinal
1.14 ng/µl
Aim:
: We need to verify that the pET43.1-C1 we isolated from transformation of the ligated products does indeed contain the C1 insert. In order to achieve that we will split the ligated vector into insert and the plasmid with restriction enzymes.
n = C*V
n = 162 * 30 = 4860 ng
We have 4860 ng and we want to digest 400 ng of DNA.
1. Add all reagent in a 1 ml Eppendorf
2. Allow the digestion to proceed during 1 h at 37°C.
3. Incubate 5 min at 65°C to inactivate the enzymes.
Aim:
We didn'tget any inserts, so we decided to repeat the preparation of pET43.1 and also the inserts C1 and C2.
What we did in the lab:
Materials:
•pET43.1 at 86.7 ng/µl,
•BamH I, Hind III, Cutsmart buffer,
•all restriction reagents being from NEB (New England Biolabs).
Method :
Prepare the mix composed of plasmid DNA, two enzymes, and buffer in an Eppendorf tube.
Table 19
pET43.1a(+) 86.7 ng/µl
Vol DNA
4.9 µl
Vol Hind III
1 µl
Vol BamH I
0.5 µl
Vol H20
11.6 µl
Vol Buffer (10X) Cutsmart
2 µl
Vol TOTAL
20 µl
Aim :
Digestion with new enzymes Xba I and Hind III of inserts (C1 and C2) and pET43.1a(+)
We have decided to switch from BamH I Hind III restriction digests to Xba I Hind III so that could remove the region containing a NusA fusion in the pET43.1a vector, as well as the following tags, and protease cleavage sites. This will make a cleaner construct for our protein.
Prepare the digestion mix for the two inserts C1 and C2, as well as for the pET43.1 vector as in table 19 below.
Proceed with the digestion for 1h30 min.
Table 20
pET43.1a(+) 130 ng/µl (3 µg)
C1 10 ng/µl (300 ng)
C2 10 ng/µl (400 ng)
Vol DNA
23 µl
30 µl
40 µl
Xba I 20 000 U/ml (10 U)
0.5 µl
0.5 µl
0.5 µl
Hind III 20 000 U/ml (20 U)
1 µl
1 µl
1 µl
Vol H20
18.5 µl
13.5 µl
3.5 µl
Vol Buffer (10X) Cutsmart
5 µl
5 µl
5 µl
Vol TOTAL
50 µl
50 µl
50 µl
1. Add all reagents in a 1 ml Eppendorf (1 for C1,1 for C2 and 1 for pET43.1)
2. Allow digestion to proceed for 1h30 at 37 °C then incubate 5 min at 65°C
In Table 20: the inserts are those synthesized by iDT
Quantity of enzymes:
Xba I:
In the meanwhile we also proceeded with some lab tasks including managing the amount of LB media that was at our disposal. We observed that repeated contaminations occurred, so we decided to aliquot the media in smaller volumes to minimize contamination of a very large volume.
- We have 500 ml of LB and we aliquot it into 10- 50 ml Falcon tubes.
The remainder of the time we proceeded with isolating the digested DNA using a gel extraction kit. But before that we needed to run a gel to separate the digested DNA from undigested one.
- We purify the inserts and pET43.1 with the QIAGEN Purification kit.
To save time, we prepared another 0.7% agarose gel that we stored at it 4°C with a wet towel containing TAE 1X (Tris-Acetate 10 mM, EDTA 1 mM).
Aim:
Determine the quantity of DNA we have after the gel extraction.
We will need to proceed with ligation of the inserts and vectors, and need to establish several ratios accurately.
What we did in the lab:
Materials:
•a 50 µl capacity quartz cuvette
Method:
We use the spectrophotometer (Ultrospec 3100 pro).
We diluted our samples: 1 µl of DNA for 49 µl of RNase free water.
We made the blank with RNase free water.
pET43.1 = 2800 µg/ml.
C1= 2700 µg/ml.
Vtaken = 1 µlof DNA
V(H2O) = 49 µl
Vfinal = 50 µl
Table 21
λ= 260 nm
pET43.1a(+) uncut
pET43.1a(+) cut
C1
C2
A260 nm
A280 nm
A260 nm /A280 nm
2.1
Cfinal(ng/µl)
2800 ng/µl
2700 ng/µl
Aim:
Check if the digestion has been correctly done, if the two restriction site of the plasmid have been digested. We used the second gel we saved to re run the digestion products, as the measurements of the spectrophotometer were inconsistent with the amount of DNA we started with !
•pET43.1a plasmid (obtained with Midiprep done 16/06/16)
•pET43.1a plasmid cut by Hind III/Xba I
•C1 and C2 cut by Hind III/Xba I
•gel 0.7% agarose
•TAE 0.5X buffer
•Electrophoresis generator at 130 V
•DNA ladder (Thermofisher Gene ruler 1 kb)
•P10 pipet, P20 pipet, test tube 250 ml, electrophoresis BIORAD Mini-Sub Cell GT, 2 types of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge Eppendorf 5415D,
Method:
1. fill the electrophoresis chamber with 0.5X TAE
2. add 10 µl of load buffer 6X in your samples to have 60 µl
3. Load samples in each lane with 20 µl of DNA
Following this deposit table:
Table 22
Lanes
L1 uncut
L2-L4 cut
L5-L7
L8-L10
L11
Name
pET43.1a(+) uncut
pET43.1a(+) X/H
C1 X/B
C2 X/B
Marker weight
DNA (µl)
50
50
50
50
50
H20 (µl)
0
0
0
0
0
Buffer T6X (µl)
10
10
10
10
10
Results:
Agarose gel (Refer to the Protocol)
L1: pET43.1 uncut
L4-L3-L2: pET43.1 digested (3 x 20 µl)
L5-L6-L7: C1 (3 x 20 µl)
L8-L9-L10: C2 (3 x 20 µl)
L11: 5 µl of ladder
Put the voltmeter at 130 V (0.3 A)
Figure 4
- We can see that for pET43.1 cut there are 2 bands (the first is pET43.1 digested partially, the second band is pET43.1 totally digested). So we get back bands of pET43.1 undigested, and we digested it again one more time.
Aim:
Get back our DNA digested and proceed with purification.
To extract DNA we use the Gel Extraction Kit from QIAGEN and we follow the different steps detailed in the kit.
Follow the table for the QG buffer volumes
Table 23
Empty Eppendorf
Eppendorf + gel
Final weight (mg)
m(pET43.1a(+) 1
1.07 g
1.52 g
0.45 mg
m(pET43.1a(+) 2
1.07 g
1.65 g
0.58 mg
m(C1)
1.07 g
1.66 g
0.58 mg
m(C2)
1.07 g
1.61 g
0.54 mg
Two lanes were used for an extraction for the pET43.1 (pET43.1(1) and pET43.1(2)). These volumes were too big to be contained in a 2 ml Eppendorf tube so we divided all in two. Finally, we had 8 tubes.
At the end of the kit, we pooled pET43.1 (1) and pET43.1 (2) together to reach 80 µl and we pooled C1 and C2. Finally, we stored only 3 tubes at -20°C.
Used Gel Extraction Kit 50 (QIAGEN)
Ex for QG volumes:
We need 3* DNA masses
pET43.1 (1)--> m = 0.45 mg
V = 3 x 0.45 = 1.3 ml = 1300 µl
Table 24
DNA weight (g)
DNA Volumes (ml)
m(pET43.1a(+) 1
0.45
1.3
m(pET43.1a(+) 2
1.58
1.8
m(C1)
0.59
1.8
m(C2)
0.54
1.6
- Volumes are too large, so we aliquot the DNA gel slices in 2 x 2 ml Eppendorf for each tubes
Table 25
DNA weight (mg)
DNA Volumes (ml)
m(pET43.1a(+) 1 x2
225
660
m(pET43.1a(+) 2 x2
790
900
m(C1) x2
295
900
m(C2) x2
270
900
- Follow KIT steps but we change step 4-6-7 and 8
- We also sub-divided the pET extraction in 2 more tubes
Step 4: for isopropanol, DNA masses are volumes of isopropanol
Ex : m(pET43.1(1)) = 225 mg V(pET43.1(1)) = 225 µl
Step 6: We wash twice (we used 750 µl of PE buffer)
Step 7: Put columns on 1.5 ml Eppendorf
Step 8: Add 20 µl of buffer EB and centrifuge it 1 min. After that, put it in the second column and add 20 µl of buffer EB.
- We have 4*20 µl tubes of pET43.1 so we regroup all of them in 1 tube. Finally, we have 1 tube for pET43.1 with 80 µl of DNA
Aim:
We want to take back the plasmid and the inserts digested.
Mixing all of reactants and let digest during 1h30 at 37 °C.
pET43.1 at 130 ng/µl will be digested by Hind III (NEB) and Xba I (NEB)
Follow table 25.
Table 26
pET43.1a(+) 130 ng/µl
Vol DNA
80 µl
Vol Hind III
15 µl
Vol Xba I
15 µl
Vol H20
0 µl
Vol buffer (10X) CutSmart
9 µl
Vol TOTAL
93 µl
Let the digestion proceed for 1H and add an extra 0.5 µl of enzyme and continue the digestion for one hour more at 37 °C.
- Place the tubes in the freezer at -20 °C because it's late.
Aim:
Determine the quantity of DNA before doing the dephosphorylation
What we did in the lab:
Method:
We use 1 µl of each sample and the Nanodrop
Table 27
C (µg/µl)
A260 nm/A280 nm
Volume (µl)
DNA (ng)/ µl
pET43.1a(+)
10.4
1.53
0.20
90.00
900.00
C1
9.00
1.48
0.04
40.00
360.00
C2
5.70
1.44
0.10
40.00
240.00
According to these results, we decided to reconcentrate the pET43.1 by using a PCR Clean-Up Kit from QIAGEN. We follow the kit steps.
• Spectrophotometer Ultrospec 3100 pro
Dilute 1/50 in TE
A260 nm=0.036
C = 90 ng/ µl
m = 4500 ng
We found that our results were strange so we use another spectrophotometer
•Spectrophotometer Ultrospec 2100 pro: blank made with TE buffer
•pET43.1 (dilution factor 400) A260 nm + 0.007, C = 140 ng/ µl
n= 40 x 140= 5600 ng
Blank made with NE and dilution 2.5/100 for the inserts C1 and C2.
C1: A260 nm= 0.009 --> C = 180 ng/µl
N = 7200 ng
C2: A260nm = 0.010 --> C= 200 ng/µl
m = 8000 ng
• Nanodrop: blank made with TE buffer
Table 28
A260 nm/A280 nm
A260 nm/A230 nm
A260 nm
A280 nm
C (ng/µl)
m (ng)
pET43.1a(+)digested on June 30
2.3
0.360
0.020
0.009
34.20
1710.00
pET43.1a(+)
1.44
0.58
0.288
0.259
19.40
2910.00
C2
1.49
0.05
0.229
0.153
11.40
456.00
C1
1.52
0.13
0.184
0.121
9.20
368.00
We pooled the two pET43.1a(+), we finally have 4620 ng of DNA in 200 µl.
We use a gel and protein purification kit Nucleospin to reconcentrate it.
We follow the kit steps and finally have 20 µl.
Then, we measure again the quantity of DNA (blank made with NE)
C = 29.5 ng/ µl so 590 ng of DNA in 20 µl.
Aim:
Avoid the self religation of the digested plasmid.
This step makes the ligation with inserts easier, as the phosphate required for forming the phosphodiester bridge is provided by the insert, therefore the reaction occurs in trans.
1. In a 1 ml Eppendorf, we put 20 µl of DNA (590 ng of pET43.1)
1 µl of rSAP (recombinant shrimp alkaline phosphatase) (diluted 1/3), 2.4 µl of buffer and 0.6 µl of H20
2. Allow the digestion to proceed during 30 min at 37°C
3. Incubate 5 min at 65°C to inactivate the enzyme
Aim:
Make the plasmid bind with the inserts before the transformation.
• Cloning efficiency competent DH5-&alpha 6)
• SOC media
• 42 °C water bath
Method:
We aliquot the competent cells in 5 tubes (80 µl per tube) and we use all our products of ligation. (refer to June 6, 2016 for detailed protocol)
The cells were incubated on ice for 30 minutes. The heat shocked for 40 secs at 42°C. They were the placed on ice for 3 min. We added 150 µl of SOC media.
We placed our tubes in the shaking incubator at 37°C and 130 RPM during 40 min. Then, we spread on petri dishes using plastic rakes, and incubated at 37°C during the night.
•pET43.1a plasmid (obtained with Midiprep done June 16, 2016)
•pET43.1a plasmid cutted by and extracted from gel 29/06/16 and digested again by same enzymes
•gel 0.7% agarose
•TAE 0.5X buffer
•Electrophoresis generator at 130 V
•DNA ladder (Thermofisher Gene ruler 1 kb)
•P10 pipet, P20 pipet, test tube 250mL, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator, centrifuge 5415D.
Method:
1. Put the gel in the electrophoresis chamber and add TAE 0.5X buffer solution to cover the gel.
2. Following this deposit table:
Table 31
Lanes
L1
L3
L5-8
Name
Marker weight
pET43.1a(+) uncut
pET43.1a(+) X/B
DNA μl
50
50
50
H2 0 µl
0
0
0
loading buffer 6X µl
10
10
10
Run at 90 V.
Molecular Weight :
- Marker Weight --> 500-1000/2000 bp molecular weight range
- Xylene cyanol dye ff --> 4000 bp (run to about this weight)
- bleu de Bromophénol--> 300 bp
- pET431A -->7200 bp
- pET43.1a--> 5200 bp
- Top of agarose gel--> 6000 bp
Results:
Agarose gel:
We can see on this second gel, pET43.1 cut there are 2 bands (the first is pET43.1 digested half, the second band is pET43.1 totally digested) again. We left the migration more longer to separate better the plasmid totally digested from the half digested one.
1. Cut agarose gel to have only the digested plasmid that we need and put it on a 2 ml Eppendorf.
Table 32
DNA weight (mg)
DNA Volumes (ml)
pET43.1a(+)
520
1.073 uncut
2. Used PCR clean-up Gel Extraction Kit (Macherey-Nagel)
NTI agarose disaggregating buffer volumes:
We need 2x DNA masses
pET43.1 (1)--> m = 520--> V = 2*520 = 1.073 ml = 1073 µl
Melting gel slices at 50°C, 10 min, separated1500 µL in 3 columns with 500 µl.
Bind DNA and remove the excess NTI buffer by centrifugation at 11000 g, 30 s.
Wash silica membrane with 700 µl of NT3 buffer at 11000 g, 30 s, twice.
Dry silica membrane 11000g for 1 min.
Elute DNA with 3 x 15 µl/Eppendorf-> final volume = 45 µl of DNA.
Aim:
We had problems to measure our DNA concentration with our spectrophotometer so we use a Nanodrop (Thermofisher)
Results:
Table 33
λ = 260nm
C1
C2
pET43.1a(+) digested by Xba I/Hind III
A260nm/A280nm
1.48
1.44
1.53
A260nm/A230nm
0.04
0.10
0.20
Cfinal (ng/µl)
9.0
5.7
10.4
• DNA reconcentration:
To dephosphorylate our vector, we need use less than 50 µl, so we reconcentrate this with the PCR clean-up Gel Extraction Kit (Macherey-Nagel).
NTI volumes:
We need 2* DNA volumes
pET43.1 (1)--> V = 90 µl --> V = 2*90 = 180 µl
Follow the kit steps
Elute in 50 µl
DNA measurements:
We try do some measurement of our vector with Eppendorf Uvette on the spectrophotometer ultrospec 3100 pro but they indicate we have more DNA than we digested. The error rate for low DNA concentration is too important with this plastic cuvette.
So we use a quartz cuvette but the error rate for low DNA concentration is too important with this cuvette too.
So we use Nanodrop
Table 34
λ = 260 nm
C1
C2
pET43.1a(+) 1 digested by Xba I/Hind III
pET43.1a(+) 2 digested by Xba I/Hind III
pET43.1a(+) control by at 120 ng/µl
A260 nm/A280 nm
1.49
1.44
1.53
1.44
1.89
A260 nm/A230 nm
0.05
0/10
0.20
0.58
1.89
Cfinal (ng/µl)
11.4
9.2
10.4
19.4
94.4
DNA quantity ng
456
368
1710
2910
So we regrouped 2 tubes of pET43.1a digested solution in one. We had 4620 ng DNA in 200 µl and we tried to reconcentrate it with the PCR clean-up Gel extraction Kit (Macherey-Nagel) in 20 µl.
• DNA reconcentration:
Incubate 30min at 37°C and inactivated 5 min at 65°C.
Ligation of pET43.1 C1 and C2:
C(pET43.1) = 29.5 ng/µl
C(C1) = 11.4 ng/µl
C(C2) = 9.2 ng/µl
- Follow the table below the volumes used.
2. Ligation:
Aim: After this third transformation without colony, we did an electrophoresis with ligation products. It is also oo check if the ligation was successful or not. This is unusual as the amount of material used is quite low. However, without proof of the ligation working we cannot ascertain many parameters, including transformaton procedures, presence of DNA, etc..
• pET43.1a plasmid (obtained with midiprep done 16/06/16)
• pET43.1a plasmid cut by Hind III/Xba I
• C1 and C2 cut by Hind III/Xba I
• gel 0.7% agarose
• TAE 0.5X buffer
• Electrophoresis generator at 130 V
• DNA ladder (Thermoscientific Gene ruler 1 kb)
• P10 pipet, P20 pipet, test tube 250 ml, electrophoresis BIORAD Mini-Sub Cell GT, 2 type of tips, 1.5 ml Eppendorf sterile tubes, 37°C water bath, shaking incubator centrifuge 5415D,
Method:
1. We redo the ligation of pET43.1 digested with C1 (refer on the June 30, 2016 for protocol) but we let the ligation run for one hour at RT.
2. We made a 0.7% agarose gel
3. Fill the electrophoresis chamber with TAE 0.5X
4. Follow this deposit table
Deposit table :
L1 --> Ladder 10 µl (Gene ruler)
L2-4 -->pET43.1 uncut (reference at 7000 bp)
L5-7 --> pET43.1 digested and dephosphorylated
L8-10 --> C1 + pET43.1 (1:0)
L11 --> C1 + pET43.1 (1:1)
L12 --> C1 + PET43.1 (1:3)
5. Put the voltmeter at 130 V during about one hour
Results:We see nothing on the gel, no DNA in our tubes. We probably made a mistake when we performed the experiment.
