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From 2010.igem.org

Contents 1 Hassan 1.1 Version 0.5.1 1.2 Version 0.6.1 2 Mimmi 2.1 pMA 2.1.1 Primer dilution 2.2 pMA/pEX 2.2.1 verification PCR 3 Andreas 3.1 Assembly of CPP⋅protein⋅His constructs 3.1.1 Transformation results 3.1.2 Colony PCR of pEX.RFP, pEX and pMA.His 3.1.2.1 Results 3.1.3 Plasmid prep 3.1.4 Assembly of His⋅SOD/yCCS into pSB1K3 3.1.4.1 Digestions 3.1.4.2 Ligations 3.1.4.3 Transformation 3.1.5 ON cultures

Hassan

midnight update, after a fast recovery from sickness!

Version 0.5.1

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Version 0.6.1

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Mimmi

pMA

Primer dilution

pMA_VF		91µl sH2O --> 100µM		10µl + 90µl sH2O --> 10µM
pMA_VR		91µl sH2O --> 100µM		10µl + 90µl sH2O --> 10µM

pMA/pEX

verification PCR

		pEX	pMA
Mix	(µl)	X6	X3		primers		conditions
sH2O	22.5	135	67.5		pEX_VF		time	°C
F primer	1	6	3		pEX_VR		2m	94
R primer	1	6	3		pMA_VF		30s	94	)
DNA	0.5	5X0.5	2X0.5		pMA_VR		30s	60	> 30 cycles
tot	25µl	250µl	75µl				2m30s	72	)
							10m	72
							oo	10

Andreas

Assembly of CPP⋅protein⋅His constructs

Continued from 19/8

Transformation results

• pEX.RFP: Good yield of red (positive) and white (negative) colonies.
• pSB1K3.SOD⋅His: Very few colonies
• pSB1K3.yCCS.His: Very few colonies
  • Km probably not feasible with quick transformation.

Colony PCR of pEX.RFP, pEX and pMA.His

Picked four pEX.RFP colonies for PCR verification. Also ran PCR on the pEX and pMA.His clones from 19/8. PCR and gel run by Mimmi (see above).

Results

• pMA.His clone verified for correct insert.
• pEX and pEX.RFP need to be re-run, as they did not result in any bands.

Plasmid prep

From 19/8 ON cultures

• E.Z.N.A. Plasmid Mini Prep kit.
• 70 μl elution volume

DNA concentrations
Sample	Conc. [ng/μl]	A260/A280
pSB1K3.BBa_J04450	103.8	1.94
pEX	55.52	1.89
pMA.His	85.67	1.87

Assembly of His⋅SOD/yCCS into pSB1K3

Step I of C-CPP cloning strategy (19/8)

Digestions

[pSB1C3.m-yCCS 1] = 101.2 ng/μl [pSB1C3.m-SOD 2] = 105.5 ng/μl

[μl]	pMA.His	pSB1C3.m-yCCS 1	pSB1C3.m-SOD 1	Inc.: 37 °C, 0:30 (FD) or 1:30 (NgoMIV)
10X FD buffer	3	3	3
dH2O	6.7†	6	7
2 μg DNA	23.3	20	19
FD EcoRI	0.5	–	–
FD PstI	–	1	1
FD AgeI	0.5	–	–
NgoMIV	–	1	1
	30†	30	30

† Miscalculation

Ligations

Without prior enzyme inactivation or DNA purification.

[μl]	Lig pSB1K3. His⋅SOD	Lig pSB1K3. His⋅yCCS	Vector/insert ratio: 1:3. Inc.: 22 °C, 0:10
100 ng vector	2.2	2.2
His insert	4.0	4.0
SOD insert	5.1	–
yCCS	–	5.7
5X Rapid Lig. buf.	4	4
dH2O	2.7	2.1
T4 DNA ligase	1	1
	20	20

Enzyme inactivation (not PstI): 80 °C, 20 min.

Transformation

Standard transformation protocol.

• 1 μl ligation mix
  • Lig pSB1K3.His⋅SOD
  • Lig pSB1K3.His⋅yCCS
• Cells plated onto 50 Km LB agar plates

ON cultures

• pMA.His
• pEX (not yet verified)
• pEX.RFP 3 (not yet verified)

3 ml LB + 100 Amp. 30 °C ON.