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From 2012.igem.org

Monday:

• PCR colony (Gibson assembly date 08.03)

• PCR run

	pBAD	PP Luc (1)	PP Luc (2)	LC Luc (1)
Nuclease-free H20	14	14	14	14
10x FD Buffer	2	2	2	2
Plasmid DNA	2	2	2	2
FD EcoRI	1	1	1	1
FD SpeI	1	0	0	0
FD XbaI	0	1	1	1

Name PCR Template Primer F Primer R Length Additive

1 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 8%Dmso
2 psigEP -LuxCDEG PCR (date 08.02) 16.H1 16.H4 4000 5%Dmso
3 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 5%Dmso
4 Pcaa3 -LuxCDEG PCR (date 08.02) 16.H9 16.H4 4000 8%Dmso
5 LuxBrick nP LuxBrick 17.E1 Suffix.Dig R 600
6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000
7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000
8 ADF-3 Alone ADF-3 17.E2 17.E3 2000
9 BBricking VB (V.fis) PSB1C3 (RS1) Suffix.Dig F Preffix.Dig R 2000
10 BBricking VB (V.fis) PSB1T3 Suffix.Dig F Preffix.Dig R 2000
11 ADF-3_VB LuxBrick 16.F7 16.F10 3000
12 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000
13 ADF-3_VB LuxBrick 16.F7 16.F10 3000
14 ADF-3+_ VB LuxBrick 16.F7 16.G2 3000

Notes

• PCR Tm=56°C.
• 11 and 12 Tm=60°C
• 13 and 14 TouchDown PCR. PCR annealing program 54°C.

Tuesday:

• PCR band recuperation

Name PCR Concentration [ng/µl] 1 and 2 psigEP -LuxCDEG 5.7

• Digestion of colonies selected in colony PCR constructs C1.1 and C1.2 (Gibson assembly date: 08.01)

C1.1: Band from colony 8 is the right size. Construct succesfully assembled and will be sequenced to confirm. C1.2 : Wrong size (all colonies). Probably due to: the use of mRFP instead of RFP for the Gibson assembly.

• Gibson results (assembly date: 08.03) and Colony PCR

C1.1: two positive colonies but were the wrong size. C1.2: several positive colonies but were the wrong size. Probably due to: the use of mRFP instead of RFP for the Gibson assembly. Pcaa3 BB: several colonies and with the right size. It will be digested. ADF 3: no colonies.

• Miniprep and positive colonies digestion (Gibson assembly date: 08.03)

Colony Concentration [ng/µl] 1 52.4 2 45.8 5 38.3 10 62.9

• Synechocystis PCC6803 transformation results (transformation date: 07.20)

Synechocystis did not grow. Probably due to: they die in the presence of glucose. Synechocystis were reinoculated and e. coli was transformed with psbAB+GFP to further transformation in synechocystis.

• PCR run (59ºC)

Name PCR Template Primer F Primer R Length Additive 1 Bactomithril 2 Bactomithril 4 Pcaa3 -LuxCDEG sigEP -LuxCDEG 16.H9 16.H4 4000 8%Dmso 5 LuxBrick (No P) LuxBrick 17.E1 Suffix.Dig R 600 5%Dmso 6 LuxBrick (wxAB) LuxBrick 17.E4 17.E5 2000 5%Dmso 7 LuxAB (4tap) LuxBrick 17.D7 17.D10 2000 5%Dmso 8 ADF-3 Alone ADF-3 17.E2 17.E3 2000 5%Dmso 9 C1_R_VB C1.1 16.E7 16.E6 4300 10 C1.2_VB C1.1 14.F6 14.F5 5200

Notes

• 9: amplifies from RS2 to mRFP. Switches Kan to KanR.
• 10: amplifies from mRFP to RS1. Switches psbAB to psbAB2 (C1.2)