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

@@ Line 1,334: / Line 1,334: @@
 <center><img src="https://static.igem.org/mediawiki/2016/5/5d/5._fig5_Pasteur.png " width="300px"; alt=""><i><p><U>Figure 5</U></p></i></center></br></br>
-<center><img src=" https://static.igem.org/mediawiki/2016/3/31/6.fig6_Pasteur.png" width="300px"; alt=""></center><i><p><U>Figure 6</U></p></i></center></br></br>
+<center><img src=" https://static.igem.org/mediawiki/2016/3/31/6.fig6_Pasteur.png" width="300px"; alt=""><i><p><U>Figure 6</U></p></i></center></br></br>
 <br /><br /><br />
              </p>

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

	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

	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

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

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

Revision as of 23:37, 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

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

	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

*Table 35*
λ = 260_nm	pET43.1a(+) digested by Xba I/Hind III
A260_nm/A280_nm	1.49
A260_nm/A230_nm	0.35
Cfinal (ng/µl)	29.5
DNA quantity	520 ng

*Table 37*
	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

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

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

Revision as of 23:37, 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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