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From 2010.igem.org

Contents 1 Nina 1.1 Agarose gel on Colony PCR 2 Andreas 2.1 Colony PCRs 2.1.1 Gel verification 2.2 PCR for Uppsala team 2.2.1 Gel verification 3 Mimmi 3.1 SOD activity 3.1.1 cultures 3.2 ON cultures 4 Johan 4.1 Miniprep 4.2 Cut miniprep 4.3 PCR screen 4.4 Gel of minipreps and PCR screen

Nina

Agarose gel on Colony PCR

I ran agarose gels on all the colony PCR screens from yesterday to check if I had inserts.

Unfortunately non of these gels show that there has been an proper insert of Fusion and Protein A all with the three CPPs in the N-terminal in the pEX vector.

I made a bad decision to conduct the ligation of these parts with the pEX vector and transform the ligations directly into overexpression cells such as BL21. I should have transformed the ligations into cloning cells such as Top 10 and from there as usual perform a mini prep and transform the vectors with insert into BL21 cells. However I hoped some of the ligations that would have ended up as vectors with insert would succesfully get transformed into BL21, but it seems from the gels that this has not happened. We are short of time in the competition and this was a test to see if I could skip the two days I had to spend with transformation into Top 10, but it unfortunatelly did not work out this time.

Andreas

Colony PCRs

• pEX.ProtA⋅His: PA 1 & 2
• pEX.IgGp: Ig 1 & 2

Standard colony PCR settings

• Elongation time: 1:20

Gel verification

Expected bands

• PA: 417 bp
• Ig: 1149 bp

Results
All four constructs confirmed.

PCR for Uppsala team

Gel verification

Expected bands

• All constructs: ≈ 6570 bp

Results
Incorrect size. Send to the Uppsala team for analysis.

Mimmi

SOD activity

-continuing

• Sonicate 3x30s at ~12amp

• Make solutions
  • WST working solution 20ml
  • Enzyme working solution 2.5ml

mix	samples	blank 1	blank 2	blank 3
sample solution	20		20
ddH2O		20		20
WST solution	200	200	200	200
Enzyme solution	20	20
dilution buffer			20	20
tot	240µl	240µl	240µl	240µl

• Incubate in 37°C for 20 min

• Measure A₄₄₀ with nanodrop

• SOD activity (inhibition %) = ((A_blank1 - A_blank3) - (A_sample - A_blank2))/(A_blank1 - A_blank3) x 100

cultures

Top10

• pEX.SOD.his.RBS.yCCS 1
• pEX.SOD.his.RBS.yCCS 1

BL21

• pEX.SOD.RBS.yCCS
• pEX.his.SOD.RBS.yCCS

ON cultures

pSB1C3.nLMWP	)
pSB1C3.nTAT	\ plasmid prep.
pSB1C3.nTra10	/
pEX.SOD.his.RBS.yCCS	)
pEX.SOD	\ SOD activity test
pEX.yCCS	/
pEX.bFGF	)
pEX.IgGp	/
pEX.Prot.A	> glycerol stock
pEX.SOD	\
pEX.yCCS	)

Johan

Miniprep

3 ml of sampled "5-6", "5-8", "AS10" and "EA3". Verification of the last constructs, his-bFGF, bFGF-his and tat-bFGF-his.

Cut miniprep

5 µl DNA

(1 µl BamHI enzyme)

2 µl 10x buffer

12 µl H2O

PCR screen

Of the other constructs in BL21 cells, tra10-bFGF-his, lmwp-bfgf-his, his-bFGF-tra10, his-bFGF-tat, his-bFGF-lmwp. 2 colonies for each plate.

0,5 µl Taq pol

0,5 µl dNTP

5 µl 5x buffer

1 µl pex for primer

1 µl pex rev primer

17 µl H2O

A mastermix 10x was made

Gel of minipreps and PCR screen

Top lanes: Cut & uncut miniprep

Bottom lanes: PCR screens

Results:

Top lanes, I realized I put BamHI even in the samples that was supposed to be uncut!

Bottom lanes: Correct size in the first lane but otherwise empty, why?