Difference between revisions of "Team:Pasteur Paris/Microbiology week4"

Aim:

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.

Protocol:

follow in this link

Aim:

Assay the DNA to check if the Midiprep was efficient, and to have the concentration of pET43.1-C1 for transformations and sequencing.

Protocol:

follow in this link

What we did in the lab:

Material:

•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 260_nm, and using ds-DNA (double stranded DNA) absorbance of 50 µg/ml for an optical density of 1 (A260_nm=1)*
λ= 260_nm	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.

Protocol:

follow in this link

What we did in the lab:

Material:

•30 µl of pET43.C1 at 162 ng/µl

Method:

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.

Protocol:

follow in this link

What we did in the lab:

Method:

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	C1
A_{260 nm}
A_{280 nm}
A260 nm /A280 nm		2.1
C_{final(ng/µl)}	2800 ng/µl	2700 ng/µl

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                              <td align = "center"; valign = "center"><strong><p> Buffer T6X (&#181;l)</p></strong></td>
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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

	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

	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

	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

Difference between revisions of "Team:Pasteur Paris/Microbiology week4"

Revision as of 23:44, 19 October 2016

click on the weeks

Microbiology Notebook

Week 4

June 27, 2016:

28. MidiPrep of pET43.1 linked with C1 (1:3)

29. Dosage of pET43.1 linked with C1 (1:3)

30. Digestion of pET43.1 linked with C1

June 28, 2016:

31. Digestion of pET43.1 linked with C1_C2

June 29, 2016:

32. Dosage of the samples digested on the June 28, 2016

33. Electrophoresis on agarose gel made on the June 28,2016

34. Extraction of the DNA from agarose gel

35. Digestion of pET43.1a (+)

June 30, 2016:

36. Dosage of DNA extracted on the June 29, 2016

June 22, 2016:

37. Dephosphorylation of the plasmid

38. Ligation of pET43.1 with C1 and C2

39. Transformation of competent cells

40. Electrophoresis of pET43.1 cut with Hind III/Xba I

41. Gel extraction

42. Nanodrop measurements

43. Measurement of DNA concentration with Nanodrop

44. Dephosphorylation of pET431.a(+) and ligation of this vector with C1 and C2

45. Transformation of bacteria with Ligation of pET43.1/ C1 and C2

July 1st, 2016:

46. Transformation of bacteria with Ligation of pET43.1/ C1 and C2

	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

	C (µg/µl)	A_{260 nm}/A_{280 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

	A_{260 nm}/A_{280 nm}	A_{260 nm}/A_{230 nm}	A_{260 nm}	A_{280 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

	1:1 (µl)	1:3 (µl)	1:0 (µl)
pET43.1a(+) dephosphorylated (100 ng)	3.5	3.5	0.0
C1/C2	1.5	1.5	0.0
T4 DNA ligase	1.0	1.0	1.0
buffer 10X	15	15	15

λ= 260_nm	C1	C2
A260 nm /A280 nm	1.71	1.80
A260 nm /A230 nm	0.39	0.39
Cfinal	9.7 ng/µl	11.0 ng/µl

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

λ = 260_nm	C1	C2	pET43.1a(+) digested by Xba I/Hind III
A260_nm/A280_nm	1.48	1.44	1.53
A260_nm/A230_nm	0.04	0.10	0.20
Cfinal (ng/µl)	9.0	5.7	10.4

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
A_{260 nm}/A_{280 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

	pET43.1a(+) (29 ng/µl)
Vol DNA(µl)	20
Vol rSAP diluted 1/3 (µl)	1
Vol H₂0 (µl)	0.6
Vol Buffer (10X) Cutsmart (µl)	2.4
Vol Total (µl)	24

	ligation ratio (insert:vector) 1:1	ligation ratio (insert:vector) 1:"	Only pET43.1a(+)
pET43.1a(+) (50 mg)	3.5	3.5	3.5
Insert (C1/C2)	1.5	1.5	0.0
T4 Ligase	1.0	1.0	1.0
Buffer 10X	1.5	1.5	1.5
H₂0	7.5	4.0	9.0
TOTAL	15.0	15.0	15.0

Difference between revisions of "Team:Pasteur Paris/Microbiology week4"

Revision as of 23:44, 19 October 2016

click on the weeks

Microbiology Notebook

Week 4

June 27, 2016:

28. MidiPrep of pET43.1 linked with C1 (1:3)

29. Dosage of pET43.1 linked with C1 (1:3)

30. Digestion of pET43.1 linked with C1

June 28, 2016:

31. Digestion of pET43.1 linked with C1_C2

June 29, 2016:

32. Dosage of the samples digested on the June 28, 2016

33. Electrophoresis on agarose gel made on the June 28,2016

34. Extraction of the DNA from agarose gel

35. Digestion of pET43.1a (+)

June 30, 2016:

36. Dosage of DNA extracted on the June 29, 2016

June 22, 2016:

37. Dephosphorylation of the plasmid

38. Ligation of pET43.1 with C1 and C2

39. Transformation of competent cells

40. Electrophoresis of pET43.1 cut with Hind III/Xba I

41. Gel extraction

42. Nanodrop measurements

43. Measurement of DNA concentration with Nanodrop

44. Dephosphorylation of pET431.a(+) and ligation of this vector with C1 and C2

45. Transformation of bacteria with Ligation of pET43.1/ C1 and C2

July 1st, 2016:

46. Transformation of bacteria with Ligation of pET43.1/ C1 and C2

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