EPF-Lausanne/25 August 2009

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(→Trp Operon synthesis)
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===Trp Operon synthesis===
===Trp Operon synthesis===
-
Using the Klenow fragments protocol and primers:£
+
Using the Klenow fragments protocol and primers:
: Trp operon Rev
: Trp operon Rev
: Trp operon Fwd
: Trp operon Fwd
-
{| class="wikitable" width="80%"
+
{|style="border:2px solid black;"  cellpadding="10" width="80%" align="center"
|+ <big> '''Trp Operon synthesis''' </big>
|+ <big> '''Trp Operon synthesis''' </big>
|-
|-
-
! scope=col |   
+
! bgcolor="#99CCFF" scope=col |   
-
! scope=col | 2x Trp  
+
! bgcolor="#99CCFF" scope=col | 2x Trp  
-
! scope=col | 1x Negative Control
+
! bgcolor="#99CCFF" scope=col | 1x Negative Control
|-
|-
| width="33%" |
| width="33%" |
NEB 2 (10x)
NEB 2 (10x)
-
| width="33%" align="center"|
+
| width="33%" align="center" |
2ul each
2ul each
-
| width="33%" align="center" |
+
| width="33%" align="center" |
2ul
2ul
|-
|-
Line 93: Line 93:
| width="34%" align="center" |
| width="34%" align="center" |
21ul
21ul
-
|----------------------------
+
|-  
-
| width="33%" |
+
|bgcolor="#CCCCFF" width="33%" |
<big>total</big>
<big>total</big>
-
| width="33%" align="center"|
+
| bgcolor="#CCCCFF" width="33%" align="center"|
-
25 ul each
+
'''25 ul each'''
-
| width="34%" align="center" |
+
| bgcolor="#CCCCFF" width="34%" align="center" |
-
25 ul
+
'''25 ul'''
 +
|-
 +
|}
 +
 
 +
The primers volumes were directly taken from the original primer dilution (iGEM 09 primers box)
 +
 
 +
'''1.''' Running Klenow 1 file on thermal cycler to denature and slowlyy anneal primers (self-annealing)
 +
'''2.''' Running Klenow file after adding the Klenow enzyme (for extension)
 +
 
 +
 
 +
===DNA preparation===
 +
For sending to sequencing
 +
{|style="border:2px solid black;"  cellpadding="1" width="33%" align="center"
 +
|-
 +
! bgcolor="#99CCFF" scope=col | sample name
 +
! bgcolor="#99CCFF" scope=col | init concentration [ng/ul]
 +
! bgcolor="#99CCFF" scope=col | final volume [ul]
 +
! bgcolor="#99CCFF" scope=col | final concentration [ng/ul]
 +
! bgcolor="#99CCFF" scope=col | sample [ul]
 +
! bgcolor="#99CCFF" scope=col | H20
 +
|-
 +
| width="33%" align="center" |
 +
BB1
 +
| width="33%" align="center" |
 +
180.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
6.94
 +
| width="33%" align="center"  |
 +
43.06
 +
|-
 +
| width="33%" align="center" |
 +
BB3
 +
| width="33%" align="center" |
 +
215.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
5.8
 +
| width="33%" align="center"  |
 +
44.2
 +
|-
 +
| width="33%" align="center" |
 +
BB5
 +
| width="33%" align="center" |
 +
350.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
3.57
 +
| width="33%" align="center"  |
 +
46.43
 +
|-
 +
| width="33%" align="center" |
 +
BB6
 +
| width="33%" align="center" |
 +
142.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
8.77
 +
| width="33%" align="center"  |
 +
41.23
 +
|-
 +
| width="33%" align="center" |
 +
RD2 #5
 +
| width="33%" align="center" |
 +
81.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
15.34
 +
| width="33%" align="center"  |
 +
34.66
 +
|-
 +
| width="33%" align="center" |
 +
LTT=LT5
 +
| width="33%" align="center" |
 +
326
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
3.83
 +
| width="33%" align="center"  |
 +
46.17
 +
|-
 +
| width="33%" align="center" |
 +
LR2
 +
| width="33%" align="center" |
 +
92
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
13.59
 +
| width="33%" align="center"  |
 +
36.41
 +
|-
 +
| width="33%" align="center" |
 +
LR5
 +
| width="33%" align="center" |
 +
47.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
26.32
 +
| width="33%" align="center"  |
 +
23.68
 +
|-
 +
| width="33%" align="center" |
 +
LR7
 +
| width="33%" align="center" |
 +
113.5
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
11
 +
| width="33%" align="center"  |
 +
39
 +
|-
 +
| width="33%" align="center" |
 +
RD2 #8
 +
| width="33%" align="center" |
 +
247
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
5.06
 +
| width="33%" align="center"  |
 +
46.94
 +
|-
 +
| width="33%" align="center" |
 +
RD2 #4
 +
| width="33%" align="center" |
 +
226
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
5.53
 +
| width="33%" align="center"  |
 +
46.47
 +
|-
 +
| width="33%" align="center" |
 +
RD2 #10
 +
| width="33%" align="center" |
 +
200
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
6.25
 +
| width="33%" align="center"  |
 +
43.75
 +
|-
 +
| width="33%" align="center" |
 +
LT2
 +
| width="33%" align="center" |
 +
141
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
8.87
 +
| width="33%" align="center"  |
 +
41.13
 +
|-
 +
| width="33%" align="center" |
 +
LT6
 +
| width="33%" align="center" |
 +
152
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
8.22
 +
| width="33%" align="center"  |
 +
41.78
 +
|-
 +
| width="33%" align="center" |
 +
LT12
 +
| width="33%" align="center" |
 +
142
 +
| width="33%" align="center"  |
 +
50
 +
| width="33%" align="center" |
 +
25
 +
| width="33%" align="center" |
 +
8.8
 +
| width="33%" align="center"  |
 +
41.2
 +
|-
 +
 
 +
|-
 +
|}
 +
 
 +
Note : BBx means LovTAP biobrick. We identified 7 clones that worked, so x is the number of the clone. RO2#x : we have 4 clones that worked for the read out 2 but the RO2#8 seems to have a weird behaviour even if we know it has the insert, so we didn't use it afterwards.
 +
 
 +
===Double transformation===
 +
We made a double transformation of RO2#4/BB1, RO2#5/BB3, RO2#8/BB5 and RO2#10/BB6.
 +
 
 +
{|style="border:2px solid black;"  cellpadding="10" width="80%" align="center"
 +
! bgcolor="#99CCFF" scope=col | 
 +
! bgcolor="#99CCFF" scope=col | RO2# [ng/ul]
 +
! bgcolor="#99CCFF" scope=col | BB [ng/ul]
 +
! bgcolor="#99CCFF" scope=col | RO2# [ng/ul]
 +
! bgcolor="#99CCFF" scope=col | BB [ng/ul]
 +
|-
 +
| width="20%" |
 +
RO2#4 / BB1
 +
| width="20%" align="center" |
 +
226
 +
| width="20%" align="center"  |
 +
180.5
 +
| width="20%" align="center"  |
 +
4.45
 +
| width="20%" align="center"  |
 +
5.55
 +
|-
 +
| width="20%" |
 +
RO2#5 / BB3
 +
| width="20%" align="center" |
 +
215
 +
| width="20%" align="center"  |
 +
215.5
 +
| width="20%" align="center"  |
 +
5
 +
| width="20%" align="center"  |
 +
5
 +
|-
 +
| width="20%" |
 +
RO2#8 / BB5
 +
| width="20%" align="center" |
 +
247
 +
| width="20%" align="center"  |
 +
350.5
 +
| width="20%" align="center"  |
 +
5.86
 +
| width="20%" align="center"  |
 +
4.14
 +
|-
 +
| width="20%" |
 +
RO2#10 / BB6
 +
| width="20%" align="center" |
 +
200
 +
| width="20%" align="center"  |
 +
142.5
 +
| width="20%" align="center"  |
 +
4.16
 +
| width="20%" align="center"  |
 +
5.84
|-
|-
|}
|}
 +
We had each time a total volume of 10ul of DNA, with the same amount of each DNA. The standard protocol was used and the cells were then spread over plates containing Kana + Amp as antibiotics.
==People in the lab==
==People in the lab==
-
Nath, Basile, Gab, Christian
+
Basile, Rafael, Nicolas

Latest revision as of 09:10, 4 September 2009



Contents

25 August 2009




Wet Lab

Miniprep:

Made according to the protocol of Aisgen Miniprep Kit Elution in 50 ul. The RD2 #5 plasmids prep as some that have already been prepared will be sent for sequencing this afternoon.


Mediums

New medium were made to grow the double-transformed DH5 a (LovTap BB and RO2) -> should be without thy.

M9 was redone, pH adjusted with amino acid and filtred. for 1L:

5g glucose
6g Na2PO4
3g KH2PO4
1g NH4Cl
0.5g NaCl
0.12g MgSO4
0.01g CaCl2


Trp Operon synthesis

Using the Klenow fragments protocol and primers:

Trp operon Rev
Trp operon Fwd
Trp Operon synthesis
2x Trp 1x Negative Control

NEB 2 (10x)

2ul each

2ul

dNTP

1ul each

1ul each

template primers

1ul (RV) + 1.5ul (FW) each

-

Klenow enzyme

1 ul each

1 ul

dH2O

18.5 ul each

21ul

total

25 ul each

25 ul

The primers volumes were directly taken from the original primer dilution (iGEM 09 primers box)

1. Running Klenow 1 file on thermal cycler to denature and slowlyy anneal primers (self-annealing) 2. Running Klenow file after adding the Klenow enzyme (for extension)


DNA preparation

For sending to sequencing

sample name init concentration [ng/ul] final volume [ul] final concentration [ng/ul] sample [ul] H20

BB1

180.5

50

25

6.94

43.06

BB3

215.5

50

25

5.8

44.2

BB5

350.5

50

25

3.57

46.43

BB6

142.5

50

25

8.77

41.23

RD2 #5

81.5

50

25

15.34

34.66

LTT=LT5

326

50

25

3.83

46.17

LR2

92

50

25

13.59

36.41

LR5

47.5

50

25

26.32

23.68

LR7

113.5

50

25

11

39

RD2 #8

247

50

25

5.06

46.94

RD2 #4

226

50

25

5.53

46.47

RD2 #10

200

50

25

6.25

43.75

LT2

141

50

25

8.87

41.13

LT6

152

50

25

8.22

41.78

LT12

142

50

25

8.8

41.2

Note : BBx means LovTAP biobrick. We identified 7 clones that worked, so x is the number of the clone. RO2#x : we have 4 clones that worked for the read out 2 but the RO2#8 seems to have a weird behaviour even if we know it has the insert, so we didn't use it afterwards.

Double transformation

We made a double transformation of RO2#4/BB1, RO2#5/BB3, RO2#8/BB5 and RO2#10/BB6.

RO2# [ng/ul] BB [ng/ul] RO2# [ng/ul] BB [ng/ul]

RO2#4 / BB1

226

180.5

4.45

5.55

RO2#5 / BB3

215

215.5

5

5

RO2#8 / BB5

247

350.5

5.86

4.14

RO2#10 / BB6

200

142.5

4.16

5.84

We had each time a total volume of 10ul of DNA, with the same amount of each DNA. The standard protocol was used and the cells were then spread over plates containing Kana + Amp as antibiotics.

People in the lab

Basile, Rafael, Nicolas



Retrieved from "http://2009.igem.org/EPF-Lausanne/25_August_2009"