EPF-Lausanne/25 August 2009

From 2009.igem.org

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|+ <big> '''Trp Operon synthesis''' </big>
 
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! bgcolor="#99CCFF" scope=col | RO2# [ng/ul]  
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Revision as of 08:53, 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

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

People in the lab

Basile, Rafael, Nicolas



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