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From 2010.igem.org

Contents 1 Hassan 2 Andreas 2.1 Assembly of SOD/yCCS⋅His constructs into pSB1K3 2.1.1 Sequencing results 2.1.2 Troubleshooting 2.1.2.1 Gel verification 2.2 Transfer of RFP cassette into pEX 2.2.1 Transformation results 2.3 Tranfer of MITF into pSB1C3 2.4 Amplification of N-CPPs 3 Mimmi 3.1 His.SOD/His.yCCS 3.1.1 plasmid mini rep 3.2 MITF-M 3.2.1 Gel 3.2.2 New transformation 3.3 pEX.RFP 3.3.1 verification PCR 3.4 CPPn 3.4.1 quick transformation

Hassan

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Andreas

Assembly of SOD/yCCS⋅His constructs into pSB1K3

Sequencing results

From 23/8 BLAST results of

pSB1K3.SOD⋅His 1 (pK-SH1)
pSB1K3.SOD⋅His 2 (pK-SH2)
pSB1K3.yCCS⋅His 2 (pK-yH2)
pSB1K3.yCCS⋅His 3 (pK-yH3)

sequences revealed that no His tag was present. In fact, although SOD and yCCS were digested with AgeI for the assembly, the insertion found between SpeI and PstI had not been removed, indicating that SOD and yCCS hadn't even been properly digested; this makes it even more puzzling how the picked clones have been to grow on Km 50 plates, as the SOD and yCCS source plasmid was pSB1C3.

The only reasonable explanation is that SOD and yCCS have been digested only by EcoRI. After enzyme inactivation, however, PstI (which cannot be heat-inactivated) has digested the genes, making them complementary for insertion into pSB1K3 target vector.

Troubleshooting

Two controls were prepared to identify the problems with SOD/yCCS⋅His cloning:

1. Gel verification of all samples digested with AgeI and EcoRI
   • Dig. pSB1K3.yCCS E+A (yCCS)
   • Dig. pSB1K3.SOD E+A (SOD)
   • Dig. pMA.His E+A (pMA)
2. Plating of sequenced clones onto both Cm 25, Km 50 and Amp 100 plates, to verify the vector resistance.
   • pSB1K3.SOD⋅His 1
   • pSB1K3.SOD⋅His 2
   • pSB1K3.His⋅SOD 1
   • pSB1K3.His⋅SOD 3

Gel verification

1 % agarose, 110 V, 40 min

Results
Fairly poor digestion of yCCS and SOD. Unclear for pMA.His, since the His fragment is too small to appear on the gel.

Transfer of RFP cassette into pEX

Transformation results

From 25/8

Good yield of both red and white colonies. Four red clones picked for colony PCR verification. PCR performed by Mimmi.

Tranfer of MITF into pSB1C3

Continued from 25/8

MITF taken over by Mimmi.

Amplification of N-CPPs

PCRs prepared from N-CPP cluster plasmid to isolate our three N-CPPs.

• N-Tra10
  • Fwd: pSB-VF2
  • Rev: pSB-VR
• N-TAT
  • Fwd: pEXf
  • Rev: pEXr
• N-LMWP
  • Fwd: pGexf
  • Rev: pGexr

Procedures according to the standard colony PCR protocol. Elongation time 0:45.

Mimmi

His.SOD/His.yCCS

plasmid mini rep

• Transfer cell culture to two 12ml falcon tubes

• Spinn down the cells at 3000rpm for 10m

• Follow the E.Z.N.A mini prep kit 1 protocol
  • Wash two times with DNA Wash buffer
  • Eluate in 50µl sH₂O

DNA concentration

His.SOD	His.yCCS
~160ng/µl	~230ng/µl

MITF-M

Gel

well	sample
1	ladder
2	MITF 1
3	MITF 2
4	Positive control

New transformation

• Follow the original protocol!

• Let the plate dry in the incubator before use

pEX.RFP

verification PCR

Mix	(µl)	X5		Primer		Conditions
sH2O	22.5	112.5		pEX_VF		time	°C
F primer	1	5		pEX_VR		2m	94
R primer	1	5				30s	94	)
DNA	0.5	5X0.5				30s	60	> 30 cycles
tot	25µl					2m	72	)
						10m	72
						oo	10

CPPn

quick transformation

• Keep on ice 5m

• Heat shock in 42°C for 30s

• Cool down on ice

• Plate on Amp-plate (dried in the incbator)