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From 2010.igem.org

Contents 1 Andreas 1.1 Assembly of new parts 1.1.1 Digestions 1.1.2 Gel verification 1.1.3 Ligations 1.1.4 Transformations 2 Mimmi 2.1 his.SOD.cTAT 2.1.1 Gel 2.1.2 PhastGel 2.2 pEX.SOD.his 2.2.1 Gel 3 Johan

Andreas

Assembly of new parts

1. pSB1K3.N-LMWP⋅SOD⋅His
   • Dig pSB1C3.N-LMWP (E+A)
   • Dig pMA.SOD⋅His (N+P)
   • Dig pSB1K3.RFP (E+P)
2. pSB1C3.N-LMWP⋅SOD⋅His
   • Dig pSB1C3.N-LMWP (A+S)
   • Dig pMA.SOD⋅His (N+S)

Digestions

	pSB1C3. N-LMWP	pMA. SOD⋅His	pSB1C3. N-LMWP	pSB1K3. N-TAT⋅SOD⋅ His 4
10X FastDigest buffer	3	3	3	2
dH2O	15.2	4.1	15.2	11.4
DNA (1 μg)	9.8	20.9	9.8	4.6
AgeI	1	0	1	0
NgoMIV	0	1	0	0
FD SpeI	1	1	0	0
FD EcoRI	0	0	1	0
FD PstI	0	0	0	1
FD XbaI	0	0	0	1
	30 μl	30 μl	30 μl	20 μl

• Incubation: 37 °C, 2:00 (NgoMIV & AgeI); 0:30 (FD)
• Inactivation: 80 °C, 20 min

Gel verification

1.5 % agarose, 120 V

Expected bands

• Dig pSB1C3.N-LMWP A+S 20/9: 2118 bp, (14 bp)
• Dig pMA.SOD⋅His N+S 20/9: 2416 bp, 503 bp
• Dig pSB1C3.N-LMWP E+A 20/9: 2063 bp, 69 bp
• Dig pSB1K3.N-TAT⋅SOD⋅His 4 X+P 20/9: ≈2200 bp, 558 bp

Results

Ligations

• [Dig pSB1K3.RFP E+P 14/9] = 66.6 ng/μl
• [Dig pMA.His⋅SOD E+A 14/9] = 66.6 ng/μl
• [Dig pSB1C3.C-TAT N+P 15/9] = 66.6 ng/μl
• [Dig pSB1C3.N-LMWP A+S 20/9] = 33.3 ng/μl
• [Dig pMA.SOD⋅His N+S 20/9] = 33.3 ng/μl
• [Dig pSB1C3.N-LMWP E+A 20/9] = 33.3 ng/μl
• [Dig pSB1K3.N-TAT⋅SOD⋅His 4 X+P 20/9] = 33.3 ng/μl

	pSB1C3. N-LMWP⋅SH	pSB1K3. N-LMWP⋅SH	pSB1A2. RBS.yCCS	pEX. N-TAT⋅SH
10X T4 Ligase buffer	2	2	2	2
dH2O	9	0	11	11
Vector DNA	2	1.5	1.5	1.5
Insert 1 DNA	6	4.5	4.5	4.5
Insert 2 DNA	–	11	–	–
T4 DNA ligase	1	1	1	1
	20 μl	20 μl	20 μl	20 μl

• Incubation: 22 °C, 15 min

Transformations

Standard transformations, procedures according to protocol.

• 1 μl ligation mix
  • Lig pSB1C3.N-LMWP⋅SH (Cm 25)
  • Lig pSB1K3.N-LMWP⋅SH (Km 50)
  • Lig pSB1A2.RBS.yCCS (Amp 100)
  • Lig pEX.N-TAT⋅SH (Amp 100 + 50 μl 0.1 mM IPTG)

Mimmi

his.SOD.cTAT

Gel

well	sample
1	ladder
2	pSB1C3.his.SOD.cTAT 1
3	pSB1C3.his.SOD.cTAT 2
4	pSB1C3.his.SOD.cTAT 3
5	pSB1C3.his.SOD.cTAT 4
6	pSB1C3.his.SOD.cTAT 5
7	pSB1C3.his.SOD.cTAT 6
8	pSB1C3.his.SOD
9	pEX.SOD

PhastGel

well	sample
1	SOD.his 0h
2	SOD.his 2h 1:1.5
3	his.SOD 0h
4	his.SOD 2h 1:2
5	yCCS 1 2h 1:2
6	ladder

pEX.SOD.his

Gel

well	sample
1	ladder
2	pEX.SOD.his
3	pEX.his.SOD
4	yCCS 1
5	yCCS 2

• Very weak bands, and in the wrong size...

Johan

• Cut tyrosinase with NgoMIV & SpeI

10 µl DNA

2 µl 10x buffer

1 µl NgoMIV

1 µl SpeI

6 µl H2O

• Cut bFGF with BamHI

2 µl DNA

2 µl 10x buffer

(1 µl BamHI)

15 µl H2O

Did a gel and showed that the bFGF are correct

• Ligated tyrosinase into pMA (vector with histag)

1 µl pMA

2 µl tyrosinase

2 µl 10x buffer

1 µl T4 ligase

14 µl H2O