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Cloning

2015/04/08

Transformation in TOP10 of pSB1K3, pSB1A3 and pET51(LS, RE)

• transformation according to protocol

2015/04/09

Repetition of transformation: pSB1A3/pSB1K3 in E.coli T10 (LS)

• Since there were no transformatin clones visible on the plate, transformation was performed again
• transformation according to protocol
• plated on LB-Amp or LB-Kan respectivly

Overnight cultures of: pET51b(+), pYM8, pSB1K3 ad pSB1A3 (JD)

• o/n culture, 5ml, 37°C: (JD)
• pET51b(+): Amp
• pYM8: Amp
• psB1K3: Kan
• psB1A3: Amp

2015/04/10

Miniprep: pEt51b(+) (from Ag Soll N-term Strep-tag+ c-term His-tag) and pYM8 (from AG Warscheid, TEV Seq. and Protein A-tag) (JD, SB, NW)

• used Peqlab kit
• concentrations:

pET51b(+)	56 ng/µl
pYM8	155 ng/µl

• psB1A3 + psB1K3: again pink colonies; even the o/n culture is pink. –> no mini (JD,SB, NW)

Glycerol stocks: pEt51b(+) and pYM8 in E.coli T10 (JD?)

• 1ml of o/n culture + 333µl 50% glycerol
• glycerol stocks in - 80°C

2015/04/11

o/n culture of pTurboYFP and pCR2.1-Avidin (JD)

• 5ml LB-Kan
• incubation at 37°C, shaking

2015/04/12

Miniprep:pTurboYFP and pCR2.1-Avidin (JD)

• glycerol stocks of both plasmids in - 80°C freezer
• miniprep with Peqlab kit
• concentrations: ?

2015/04/14

Primer design for pIg15_001, pIG15_002 and pIG15_101 (RE)

• pIG15_001: prokaryotic expression vector backbone
• pIG15_002: eukaryotic expression vector backbone
• pIG15_101: pET51b+ with YFP
• ordered from Sigma-Aldrich
• delivery expected at 2015/04/17

2015/04/15

Primer design for cell-free expression vector backbones and pIG15_102 (RE)

• pIG15_102: pET51b+ with GFP
• ordered from Sigma-Aldrich
• delivery expected at 2015/04/20

Mini-Prep: pET22b(+) and paFITC-C (LS, RE)

• used Peqlab kit
• concentrations:

pET22b(+)	74 ng/µl
paFITC-C	307,4 ng/µl

Autoclaved tips, eppis, beads

2015/04/16

o/n culture for pET22b+ and paFITC-C (RE)

• 5ml liquid LB
• incubation at 37°C, shaking

2015/04/17

Glycerin stocks for pET22b+ and paFITC-C (?)

• stored at -80°C (Box: iGEM Gly-stocks)

Obtained competent E. coli BL21, DE3 pLysS and Arctic RIL from Ernst Aichinger (2 aliquots each)(RE,LS)

2015/04/18

o/n culture of Arctic RIL, Rosetta and BL21 p-Lys prepared (LS)

• 2x5ml each
• incubation at 37°C, shaking

2015/04/19

Preparation of competent cells (JD,LS,RE)

• competent cells prepared according to the protocol „competent cells (new)“
• E. coli Bl21 DE3 pLysS and E. coli Rosetta
• 80 aliquots à 50 µl of each strain
• stored at -80°C

2015/04/20

Transformatio: pSB1C3 SEAP RFC10 2.2 (2014) in E. coli TOP10 (Cml) (RE)

• according to the protocol

2015/04/21

iGEM Support Kit by NEB delivered

• enzymes, buffers and DNA ladder stored at -20°C (1) in a seperate box
• competent cells stored at -80°C in a seperate box

2015/04/27

Primers arrived

• Primer Dilution according to manufacturer's instructions
• Primer oIG15_001-oIG15_20
• Primer oIG15_101-oIG15_104
• Primer stocks are in the -20°C freezer 1 in box: Primer Stock
• Primer dilutions (1:10) are in the -20°C freezer as well in box: Primer Dilutions.

2015/04/28

PCR for construction of pIG15 (?)

• oIG15_001 + oIG15_002 (pet22b+) –> LacI
• oIG15_003 + oIG15_004 (paFITC) –> CMV
• oIG15_005 + oIG15_006 (paFITC) –> WPRE
• oIG15_007 + oIG15_008 (pet22b+) –> T7 P
• oIG15_009 + oIG15_010 (pet22b+) –> T7 T
• oIG15_101 + oIG15_102 (pTYFP) –> TYFP

• MasterMix (NEB protocol): ?

• PCR program:

step	temperature [°C]	time[s]
initial denaturation	98	180
denaturation	98	10
annealing	60	20
extension	72	25
final extension	72	600

• PCR product stored in the freezer
• prepared 1% agarose gel (1x TAE buffer used for running the gel)
• 110V, 55min

• first reaction didn't work–> no lacI (lacI was the biggest fragment, so this is could be an explanation)
• the next 4 reaction resultet in a fragment with the right size
• last reaction seems to be to big, we expected ~700bp.

gel picture:

---

Gel extraction (Qiagen kit)

• elution in 50 µl elution buffer
• stored PCR gel-ex in -20°C

2015/04/29

NanoDrop analysis of the extracted PCR products (?)

fragment	concentration [ng/µl]
CMV promoter	9.3
WPRE	23.4
T7 promoter	9.2
T7 terminator	12.2
TurboYFP	10.9

• repetition of PCR from yesterday in 50 µl scale (instead of 20 µl)
• changes in cycling conditions: (initial) denaturation temp = 92°C; denaturation time = 20 s

• 1% agarose gel with 1xTAE buffer:
• 140V, 30 min
• 3 µl of every PCR product

[gel pic]

• amplification of every fragment was successful!