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From 2010.igem.org

Contents 1 Andreas 1.1 Cloning of SOD into pMA.His 1.1.1 Transformation results 1.1.2 Colony PCR 1.1.3 Gel verification 1.1.4 ON cultures 1.2 N-CPP extraction 1.2.1 Gel extraction 1.2.2 Gel verification 1.3 Cloning of N-CPPs into pSB1C3 1.3.1 Digestion 1.3.1.1 Dephosphorylation 1.3.2 Ligation 1.3.3 Transformation 2 Mimmi 2.1 MITF-M 2.1.1 Colony PCR 2.1.2 Gel

Andreas

Cloning of SOD into pMA.His

Transformation results

From 28 28/8 Good colony yield. Four colonies (SH1-SH4) picked for colony PCR.

Colony PCR

• SH1-SH4: pMA.SOD⋅His
• PC: Positive control; pMA.His
• NC: Negative control; blank

Procedures according to standard colony PCR protocol. Elongation time 1:00.

Gel verification

1 % agarose, 100 V

Expected bands:

• pMA.SOD⋅His: 831 bp
• pMA.His: 348 bp

Results
Well corresponding bands indicating successful insertion of SOD into the vector.

ON cultures

SH1 and SH2 selected for plasmid prep and sequencing. Set 5 ml LB + 100 Amp ON cultures. 37 °C, 225 rpm.

N-CPP extraction

Gel extraction

From 28/8 samples Purification using the E.Z.N.A. Gel Extraction kit. Elution in 30 μl dH₂O; double elution.

† Phenolate contaminated sample? High absorbtion at 230 nm [http://en.wikipedia.org/wiki/Nucleic_acids_analysis#Other_common_contaminants]

DNA concentrations
Sample	Conc. [ng/μl]	A260/A280
Tra10	13.56	1.86
TAT	1.736	1.20
LMWP †	2.523	2.69

Gel verification

Ran a gel to verify that the presence and size of our extracted DNA fragments.

1 % agarose, 100 V

Results
Weak band for Tra10, no bands for TAT and LMWP. Proceeded to cloning anyway.

Cloning of N-CPPs into pSB1C3

A last-minute decision was made to also make a bulk cloning of all three N-CPPs by digesting directly from the N-CPP cluster vector.

Digestion

[N-CPP plasmid]=672 ng/μl (28/8)

[ng/μl]	Tra10	TAT	LMWP	N-CPP
10X FD buffer	3	3	3	3
dH2O	3	3	8	23
FD XbaI	0.5	0.5	0.5	0.5
FD AgeI	0.5	0.5	0.5	0.5
DNA	23	23	18	3
	30	30	30	30

Incubation: 37 °C, 30 min
Inactivation: 80 °C, 10 min

Dephosphorylation

Treated the N-CPP sample with FastAP alkaline phosphatase to prevent multiple insertions (3, 5, etc...) into target vector.

• 3 μl FastAP
  • Incubation: 37 °C, 10 min
• Inactivation at 75 °C, 5 min

Ligation

[Dig. pSB1X3 X+A EXTR]=13.72 ng/μl (digested and extracted vector from 9/8) [Dig. N-CPP X+A]=60 ng/μl

[ng/μl]	N-CPP	Tra10	TAT	LMWP
Vector DNA	3	3	3	3
Insert DNA	9	12	12	12
5X Rapid Lig. buf.	4	4	4	4
dH2O	3	0	0	0
T4 DNA Ligase	1	1	1	1
	20	20	20	20

Transformation

Standard transformation protocol.

• 2 μl ligation mix
• Cm 25

Mimmi

MITF-M

Colony PCR

• Made by Andreas --> ~280bp bands, empty vector? should not be possible...

• Try again, more colonies

Mix	(µl)	X8		Primers		Conditions
sH2O	22.5	180		pSB1_VF2		time	°C
F primer	1	8		pSB1_VR		2m	94
R primer	1	8				30s	94	)
DNA	0.5	8x0.5				30s	50	> 30 cycles
tot	25µl					2m40s	72	)
						10m	72
						oo	10

Gel

well	sample
1	ladder
2	pSB1C3.MITF-M 1
3	pSB1C3.MITF-M 2
4	pSB1C3.MITF-M 3
5	pSB1C3.MITF-M 4
6	pSB1C3.MITF-M 5
7	pSB1C3.MITF-M 6
8	pSB1C3.MITF-M 7
9	blank

• No product, trying another 7 colonies over night...