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From 2010.igem.org

Contents 1 Andreas 1.1 Cloning of N-CPPs into pSB1C3 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.4 ON cultures 1.1.5 PCR product digestion 1.1.6 Gel verification 1.2 Transfer of m-yCCS into pEX 1.2.1 ON cultures 2 Mimmi 2.1 SOD.his / his.SOD 2.1.1 col-PCR 2.1.2 gel 2.2 MITF-M 2.2.1 Digestion 2.2.2 Gel 2.2.3 Ligation (nr3) 2.2.4 Transformation

Andreas

Cloning of N-CPPs into pSB1C3

Transformation results

From 6/9 transformations

Good colony yields on both plates, 1 and 2*.

Colony PCR

4 colonies picked from each plate for verification by colony PCR.

• 1, 2, 3, 4, 5*, 6*, 7*, 8*
• PC: pSB1C3.RFP

PCR tubes
dH2O	16.22
DreamTaq buffer	2
dNTP, 10 mM	0.4
Fwd primer (VF2)	0.4
Rev primer (VR)	0.4
Cell suspension	0.5
DreamTaq pol.	0.08
	20 μl

Standard colony PCR settings

• Elongation time: 0:45

Gel verification

1.5 % agarose, 90 V.

Expected bands

• pSB1C3.Tra10: 389 bp
• pSB1C3.TAT: 359 bp
• pSB1C3.LMWP: 368 bp

Results
Relevant bands in clones 3, 4, 6, 7 & 8. Slightly different in size, which could indicate presence of all three N-CPPs. All clones selected for sequencing.

ON cultures

For plasmid prep

• pSB1C3.N-CPP: pC.NCPP 3, 4, 6*, 7* and 8*
  • 5 ml LB + Cm 25
  • 37 °C, 200 rpm ON

PCR product digestion

From 6/9 PCR

10X Buffer Tango	5
PCR DNA	40
dH2O	0
AgeI	2
XbaI	1
	50 μl

• Incubation: 37 °C, 3 h
• Inactivation: 80 °C, 20 min

Gel verification

1.5 % agarose, 90 V

Expected bands

• Tra10: 182 bp
• TAT: 91 bp
• LMWP: 94 bp

Results
Very irregular band sizes in the "lower" region, making no sense. Sizes not at all corresponding to what was expected.
Higher up very regular bands. This is even more puzzling, since different primer pairs were used for each PCR reaction. Although concentration of plasmid should be too low, the bands seen may be undigested and digested N-CPP plasmid. A too large plasmid concentration may in turn be the cause of the unspecific bands seen lower down in the gel.

Transfer of m-yCCS into pEX

ON cultures

Set ON cultures of pEX.yCCS for plasmid prep and glycerol stocks. From 3/9 colonies.

• pEX.yCCS 5 and 8
  • 5 ml LB + Amp 100
  • 37 °C, 200 rpm ON
• pEX.yCCS 5 and 8
  • 5 ml LB + Amp 100
  • 30 °C

Mimmi

SOD.his / his.SOD

col-PCR

mix	(µl)	x9 x2		primers		conditions
sH2O	13.5	121.5		pSB_VF2		time	°C
dNTP	2	18		pSB_VR		2m	95
F primer	0.8	7.2		&		30s	95	)
R primer	0.8	7.2		pEX_VF		30s	55	> 30 cycles
buffer	2	18		pEX_VR		1m45s	72	)
polymerase	0.4	3.6				10m	72
DNA	0.5					OO	10
tot	20µl	180µlx2

gel

well	sample	well	sample
1	ladder	12	ladder
2	pSB1C3.SOD.his 1	13	pEX.SOD.his 1
3	pSB1C3.SOD.his 2	14	pEX.SOD.his 2
4	pSB1C3.SOD.his 3	15	pEX.SOD.his 3
5	pSB1C3.SOD.his 4	16	pEX.SOD.his 4
6	pSB1C3.his.SOD 1	17	pEX.his.SOD 1
7	pSB1C3.his.SOD 2	18	pEX.his.SOD 2
8	pSB1C3.his.SOD 3	19	pEX.his.SOD 3
9	pSB1C3.his.SOD 4	20	pEX.his.SOD 4
10	positive control	21	blank

• Nothing...

MITF-M

Digestion

Mix	(µl)		(µl)		Conditions
DNA	40		10		Time	°C
10x buffer	5		5		30m	37
sH2O	3		4		20m	65
EcoRI	1	PstI	1		oo	10
SpeI	1		0
tot	50µl		20µl

Gel

well	sample
1	ladder
2	MITF-M
3	MITF-M
4	MITF cut E+S
5	MITF cut P
6	pSB1C3 cut E+S
7	pSB1C3.MITF lig
8	pSB1C3.MITF lig

Ligation (nr3)

mix	(µl)		[pSB1C3]~100ng/µl		conditions
pSB1C3	2				time	°C
MITF-M	13				10m	22
5xbuffer	4
T4 ligase	1
sH2O	0
tot	20µl

Transformation

• Follow original protocol