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From 2010.igem.org

Contents 1 Andreas 1.1 Transfer of yCCS into pEX 1.1.1 Glycerol stocks 1.1.2 Plasmid prep 1.2 Cloning of N-CPPs into pSB1C3 1.2.1 Plasmid prep 1.2.2 Sequencing 1.3 Construction and cloning of MITF 1.3.1 MITF PCR 1.3.2 Gel verification 1.4 Colony PCRs 1.4.1 Gel verification 1.4.2 ON cultures 2 Hassan 2.1 Animation

Andreas

Transfer of yCCS into pEX

Glycerol stocks

From 7/9 ON cultures

• pEX.yCCS 5 2010-09-08
• pEX.yCCS 8 2010-09-08

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample	Conc [ng/μl]	A260/A280
pEX.yCCS 5	56.32	2.02
pEX.yCCS 8	57.63	1.97

Cloning of N-CPPs into pSB1C3

Plasmid prep

From 7/9 ON cultures
Elution: 70 μl x2

Sample	Conc [ng/μl]	A260/A280
pSB1C3.N-CPP 3	105.6	1.94
pSB1C3.N-CPP 4	88.87	2.03
pSB1C3.N-CPP 6*	86.64	1.98
pSB1C3.N-CPP 7*	75.79	1.90
pSB1C3.N-CPP 8*	52.64	1.99

Sequencing

15 μl plasmid DNA + 1.5 μl VF2 primer.

• NCPP 3: ASB0045 B41
• NCPP 4: ASB0045 B42
• NCPP 6: ASB0045 B43
• NCPP 7: ASB0045 B44
• NCPP 8: ASB0045 B45

Construction and cloning of MITF

MITF PCR

PCR tubes (MITF 1 & MITF 2)
dH2O	41
Pfu buffer	5
dNTPs, 10 mM	1
Fwd primer (MITF_FB_F_18aug)	1
Rev primer (MITF_FB_R_18aug)	1
Template DNA (pRC/CMV MITF-M, 10 ng/μl)	0.5
Pfu polymerase	0.5
	50 μl

PCR settings
95 °C	10:00
95 °C	0:30	x5
55 °C	0:30
72 °C	1:40
95 °C	0:30	x25
72 °C	2:10
72 °C	10:00

Gel verification

1 % agarose, 110 V

• 3 μl DNA ladder (O'GeneRuler 1 kb DNA ladder
• 5 μl sample

No gel image available

Results
No visible bands for neither of the two MITF samples.

Colony PCRs

Picked colonies from Mimmi's plates:

• pSB1C3.MITF (2/9)
  • MITF 1-8
• pSB1C3.SOD⋅His (6/9)
  • pC.SH 1-3
• pSB1C3.His⋅SOD (6/9)
  • pC.HS 1-3
• pEX.His⋅SOD (6/9)
  • pEX.SH 1-3
• pEX.SOD⋅His (6/9)
  • pEX.HS 1-3

Primers

• pSB1C3: VF2 & VR
• pEX: pEXf & pEXr

PCR tubes
dH2O	16.22
DreamTaq buffer	2
dNTPs, 10 mM	0.4
DreamTaq pol.	0.08
Fwd primer	0.4
Rev primer	0.4
Cell susp. (template)	0.5
	20 μl

PCR settings
95 °C	3:00
95 °C	0:30	x30
55 °C	0:30
72 °C	1:00
72 °C	5:00

Gel verification

1 % agarose, 90 V, 50 min

Expected bands

• MITF: 1586 bp
• pC.SH/pC.HS: 815 bp
• pEX.SH/pEX.HS: 702 bp

Results
All MITF samples resulted in ≈300-350 bp bands, which corresponds to re-ligated pSB1C3 vector (316 bp). This is strange, since re-ligation should not be possible.
All other samples correspond very well to expected sizes. pC.SH3, pC.HS1, pEX.SH1 & pEX.HS1 were selected for plasmid prep and glycerol stocks.

ON cultures

• 5 ml LB + 25 Cm; 37 °C, 225 rpm
  • pC.SH3
  • pC.HS3
• 5 ml LB + 100 Amp; 37 °C, 225 rpm
  • pEX.SH1
  • pEX.HS1
• 3 ml LB + 25 Cm; 30 °C
  • pC.SH3
  • pC.HS3
• 3 ml LB + 100 Amp; 30 °C
  • pEX.SH1
  • pEX.HS1

Hassan

Animation