"

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 Transformation results 3.1.1 Colony PCR of pEX.RFP, pEX and pMA.His 3.1.1.1 Results 3.1.2 ON cultures 3.2 Plasmid prep 3.3 Assembly of His⋅SOD/yCCS into pSB1K3 3.3.1 Digestions 3.3.2 Ligations 3.3.3 Transformation 4 Nina 4.1 Colony PCR on fusion protein 4.2 Agarose gel on fusion protein 4.3 Overnight culture of fusion protein

Hassan

midnight update, after a fast recovery from sickness!

Version 0.5.1

---

Version 0.6.1

---

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

Transformation results

From 19/8 transformations

• 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.

ON cultures

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

3 ml LB + 100 Amp. 30 °C ON.

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

---

Nina

Colony PCR on fusion protein

I performed a colony PCR on the dish with fusion proteins.

PCR mix for 5 tubes:

• MgCl 5 ul
• Buffer 5X 50 ul
• dNTP 5 ul
• primer revers for IgG protease 15 ul
• primer VF2 15 ul
• polymerase PjuX7 5 ul
• H2O 150 ul

PCR prgm:

Agarose gel on fusion protein

I ran the PCR product on an 1 % agarose gel 100 V in order to verify that I have bands corresponding to a correct size of the fusion protein.

Ladder: GeneRuler™ DNA Ladder Mix, ready-to-use, 100-10,000 bp Fermentas

Arrangement on gel:

Lane nr 3 looks like it could represent the wanted band of the fusion protein by protein A and IgG protease, which could be about 1300 nt.

Overnight culture of fusion protein

I inoculated colony nr 3 of the dish with the fusion protein into 12 ml LB and 24 ul chloramphenicol (50 mg/ml). This was incubated in 37 °C in shake overnight.