EPF-Lausanne/25 August 2009

From 2009.igem.org

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The primers volumes were directly taken from the original primer dilution (iGEM 09 primers box)
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'''1.''' Running Klenow 1 file on thermal cycler to denature and slowlyy anneal primers (self-annealing)
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'''2.''' Running Klenow file after adding the Klenow enzyme (for extension)
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===DNA preparation===
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For sending to sequencing
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{|style="border:2px solid black;"  cellpadding="10" width="80%" align="center"
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|-
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! bgcolor="#99CCFF" scope=col | sample name
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! bgcolor="#99CCFF" scope=col | init concentration [ng/ul]
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! bgcolor="#99CCFF" scope=col | final volume [ul]
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! bgcolor="#99CCFF" scope=col | final concentration [ng/ul]
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! bgcolor="#99CCFF" scope=col | sample [ul]
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! bgcolor="#99CCFF" scope=col | H20
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|-
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| width="33%" align="center" |
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BB1
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| width="33%" align="center" |
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180.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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6.94
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| width="33%" align="center"  |
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43.06
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|-
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| width="33%" align="center" |
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BB3
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| width="33%" align="center" |
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215.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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5.8
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| width="33%" align="center"  |
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44.2
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|-
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| width="33%" align="center" |
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BB5
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| width="33%" align="center" |
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350.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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3.57
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| width="33%" align="center"  |
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46.43
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|-
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| width="33%" align="center" |
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BB6
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| width="33%" align="center" |
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142.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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8.77
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| width="33%" align="center"  |
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41.23
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| width="33%" align="center" |
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RD2 #5
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| width="33%" align="center" |
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81.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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15.34
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| width="33%" align="center"  |
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34.66
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|-
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| width="33%" align="center" |
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LTT=LT5
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| width="33%" align="center" |
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326
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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3.83
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| width="33%" align="center"  |
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46.17
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|-
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| width="33%" align="center" |
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LR2
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| width="33%" align="center" |
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92
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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13.59
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| width="33%" align="center"  |
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36.41
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|-
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| width="33%" align="center" |
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LR5
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| width="33%" align="center" |
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47.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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26.32
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| width="33%" align="center"  |
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23.68
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|-
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| width="33%" align="center" |
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LR7
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| width="33%" align="center" |
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113.5
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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11
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| width="33%" align="center"  |
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39
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|-
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| width="33%" align="center" |
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RD2 #8
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| width="33%" align="center" |
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247
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| width="33%" align="center"  |
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50
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| width="33%" align="center" |
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25
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| width="33%" align="center" |
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5.06
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| width="33%" align="center"  |
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46.94
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==People in the lab==
==People in the lab==

Revision as of 12:28, 2 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



People in the lab

Nath, Basile, Gab, Christian



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