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From 2010.igem.org

Contents 1 Hassan 2 Andreas 2.1 Troubleshooting of SOD⋅His fusion fail 2.1.1 Restreak results 2.2 Transfer of SOD into pMA.His (pMA.SOD⋅His) 2.2.1 Strategy 2.2.2 Digestion 2.2.2.1 Gel verification 2.2.3 Gel extraction 2.3 Isolation of CPPs from N-CPP cluster 2.3.1 Gel verification 2.3.2 Gel extraction 3 Mimmi 3.1 MITF-M 3.1.1 re-digest and ligate 3.1.2 Transformation 3.2 pEX.RFP & CPPn 3.2.1 Gel 3.2.2 pEX.RFP plasmid mini prep 3.2.3 CPPn culture and glycerol stock

Hassan

added some text, animation is also a little slower now, a border added.

other things to be changed now are:

• a darker color for after pigmentation.
• colors need to be changed for proteins and genes.
• a more proper timing for after pigmentation.

Andreas

Troubleshooting of SOD⋅His fusion fail

Restreak results

From 26/8

All restreaked clones (pSB1K3.SOD⋅His 1 & 2 and pSB1K3.His⋅SOD 1 & 3) were able to grow on both Cm 25 and Km 50. This raises the question whether our plasmid actually carries resistance to both Km and Cm. To test this further, new restreaks were made, where colonies from the Cm 25 plate were streaked on a new Km 50, and vice versa. Also, pSB1K3.RFP, pSB1C3.RFP and untransformed Top10 cells were streaked.

• Amp 100 plate
  • pSB1K3.SOD⋅His 1 from Km 50 plate
  • pSB1K3.His⋅SOD 1 from Km 50 plate
  • Top10
  • pSB1K3.RFP
  • pSB1C3.RFP
• Cm 25 plate
  • pSB1K3.SOD⋅His 1 from Km 50 plate
  • pSB1K3.His⋅SOD 1 from Km 50 plate
  • Top10
  • pSB1K3.RFP
  • pSB1C3.RFP
• Km 50 plate
  • pSB1K3.SOD⋅His 1 from Cm 25 plate
  • pSB1K3.His⋅SOD 1 from Cm 25 plate
  • Top10
  • pSB1K3.RFP
  • pSB1C3.RFP

Transfer of SOD into pMA.His (pMA.SOD⋅His)

A new strategy attempted for fusing SOD to His. This time SOD will be transfered to pMA carrying the His-tag, thereby avoiding cloning/excising the small tag from its vector.

Strategy

• pMA.SOD⋅His
  • SOD digested with EcoRI and AgeI
  • pMA.His digested with EcoRI and NgoMIV
• pMA.His⋅SOD
  • SOD digested with NgoMIV and PstI
  • pMA.His digested with AgeI and PstI

Digestion

[pMA.His]=85.67 ng/μl
[pSB1C3.SOD]=105.5 ng/μl

	pMA.His	pSB1C3.SOD
10X FD buffer	2	2
DNA (1 μg)	11.7	9.5
dH2O	4.3	6.5
FD EcoRI	1	1
NgoMIV	1	0
FD AgeI	0	1
	20	20

Incubation

• FastDigest enzymes: 37 °C, 30 min
• NgoMIV: 37 °C, 2 h

Gel verification

• Dig.SOD E+N (SOD)

1% agarose, 100 V, 45 min

Results
Gel shows successful digestion: no undigested plasmid and a band corresponding well to the expected SOD size (492 bp).

Gel extraction

Remaining 22 μl SOD sample run on 1 % agarose gel, 100 V. SOD DNA band excised (240 mg gel; "Extr. Dig SOD E+A") and saved in -20 °C until tomorrow.

Isolation of CPPs from N-CPP cluster

Gel verification

Gel verification of Nina's CPP cluster PCR

1.5 % agarose, 100 V, 25 min
Expected band: 379 bp

Gel extraction

≈ 35 μl sample. CPP cluster band excised (220 mg gel; "Extr. N-CPP clust") and saved in -20 °C.

Mimmi

MITF-M

re-digest and ligate

					Conditions
Mix	(µl)				time	°C
DNA	10		DNA = pSB1C3.RFP		10m	37
sH2O	15		+		20m	65
10x buffer	3		MITF-M		oo	10
EcoRI	1
SpeI	1		( [pSB1C3]=50ng/µl, [MITF-M]=160ng/µl )
tot	30µl

• Ligate

Mix	(µl)		Conditions
cut pSB1C3	3		time	°C
cut MITF-M	3		10m	22
5x buffer	4		oo	4
T4 ligase	1
sH2O	9
tot	20µl

Transformation

• Follow original protocol

pEX.RFP & CPPn

Gel

well	sample
1	1kb ladder
3	pEX.RFP 1
4	pEX.RFP 2
5	pEX.RFP 3
6	pEX.RFP 4
7	pEX blank
8	Transportan10
9	TAT (dowdy's)
10	LMWP
11	50bp ladder

pEX.RFP plasmid mini prep

• PEX_VR binds perfectly to a region in RFP and pEX_VF binds with two mismatches to a region in RFP, so 4 different bands could be expected:
  • 1272bp
  • 1124bp
  • 1010bp
  • 862bp
• Two clear bands and one faint band obtained in the colony-PCR in the right sizes.

CPPn culture and glycerol stock

• Pick one colony from the transformation plate and add 7ml LB with Amp

• 1600µl culture in pre-sterile glycerol

• Leave the rest of the culture ON for plasmid prep