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From 2010.igem.org

Contents 1 Mimmi 1.1 MITF-m 1.1.1 Gel 1.1.2 Re-ligation and transformation 2 Andreas 2.1 Cloning of SOD into pMA.His 2.1.1 Plasmid prep 2.1.2 Sequencing 2.2 N-CPP cloning 2.2.1 Transformation results 2.2.2 Colony PCR 2.2.3 Gel verification 2.2.4 Troubleshooting/Gel verification 2.3 LB agar plates

Mimmi

MITF-m

Gel

well	sample
1	ladder
2	pSB1C3.MITF-M 8
3	pSB1C3.MITF-M 9
4	pSB1C3.MITF-M 10
5	pSB1C3.MITF-M 11
6	pSB1C3.MITF-M 12
7	pSB1C3.MITF-M 13
8	pSB1C3.MITF-M 14
9	positive control
10	digested MITF (E+S)
11	digested pSB1C3 (E+S)

Re-ligation and transformation

• Depohosphorylate vector:

Mix			Conditions
cut pSB1C3	9		time	°C
A phosp	1		10m	37
tot	10µl		5m	75
			oo	10

• Ligate

Mix	(µl)		Conditions
cut pSB1C3	2		time	°C
cut MITF-M	13		10m	22
5x buffer	4
T4 ligase	1
sH2O	0
tot	20µl

• Follow the original transformation protocol

Andreas

Cloning of SOD into pMA.His

Plasmid prep

Sample	Conc [ng/μl]	A260/A280
pMA.SOD⋅His 1	246.3	1.89
pMA.SOD⋅His 2	230.7	1.87

From 30/8 ON cultures

• E.Z.N.A. Plasmid Miniprep kit
• Elution: 50 μl dH₂O (x2)

Sequencing

pMA.SOD⋅His 1 (SH1): ASB0045 B28 pMA.SOD⋅His 2 (SH2): ASB0045 B29

N-CPP cloning

Transformation results

• pSB1C3.Tra10
• pSB1C3.TAT
• pSB1C3.LMWP
• pSB1C3.N-CPP

A dozen colonies on each plate

Colony PCR

Due to lack of PCR beads only two colonies from each single N-CPP was picked for colony PCR:

• Tra10: 10 1 & 10 2
• TAT: TA 1 & TA 2
• LMWP: LM 1 & LM 2
• Neg. contr (NC): Blank

Standard colony PCR procedures. Elongation time: 1:00

Gel verification

1 % agarose, 110 V

Expected bands

• Tra10: 389 bp
• TAT: 359 bp
• LMWP: 368 bp

Results
Way too large bands, which is very strange. Troubleshooting needed.

Troubleshooting/Gel verification

Ran a gel of N-CPP ligation mixes and digestion samples to verify DNA fragment lengths.

Expected bands

• Lig Tra10 (Lig pSB1C3.Tra10 30/8)
  • 2145 bp, 54 bp, 2078 bp
• Lig TAT (Lig pSB1C3.TAT 30/8)
  • 2115 bp, 45 bp, 2078 bp
• Lig LMWP (Lig pSB1C3.LMWP 30/8)
  • 2124 bp, 75 bp, 2078 bp
• Lig N-CPP (Lig pSB1C3.N-CPP 30/8)
  • 2124 bp, 2115 bp, 2145 bp, 54 bp, 45 bp, 75 bp, 2078 bp etc.
• Dig X+A (Dig pSB1C3 X+A EXTR (from 9/8))
  • 2078 bp
• Dig N+S (Dig pSB1C3 N+S EXTR (from 9/8))
  • 2076 bp

Results
As expected a 2078/2076 bp digested pSB1C3 fragment appeared in all samples. Apart from the ≈4 kb fragment in "Lig N-CPP" no bands are visible. This could mean that no other sizes are present; however, since almost all expected bands were very small, it is also possible that they can't be visualized by gel verification. Not many conclusions can be drawn from this.

LB agar plates

Prepared 20 x LB agar plates with 25 μg/ml Cm.