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Revision as of 12:55, 30 August 2010 (view source) AndreasConstantinou (Talk | contribs) (→Amplification of N-CPPs from N-CPP cluster) ← Older edit		Latest revision as of 11:03, 26 October 2010 (view source) Hassanforoughiasl (Talk | contribs)
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Latest revision as of 11:03, 26 October 2010

Contents 1 Andreas 1.1 Gel extraction 1.2 DNA purification of "Dig. pMA.His (E+N)" 1.3 Gel verification 1.3.1 Results 1.4 Cloning of SOD into pMA.His 1.4.1 Ligation 1.4.2 Transformation 1.5 Amplification of N-CPPs from N-CPP cluster 1.5.1 Gel verification 1.5.1.1 Results 1.5.2 Gel extraction 1.6 N-CPP 1.6.1 Plasmid prep 1.6.2 Glycerol stock

Andreas

Gel extraction

No measurable DNA conc. for "Extr. Dig SOD E+A"

DNA concentrations
Sample	Conc [ng/μl]	A260/A280
Extr. N-CPP	40.91	1.69
Extr. Dig SOD E+A	–	–
Pur. Dig pMA.His E+N	36.02	1.80

From 27/8
Purified DNA from excised samples "Extr. Dig. SOD E+A" and "Extr. N-CPP cluster" using the E.Z.N.A. Gel extraction kit; procedures according to provided protocol.

Elution volume: 30 μl (eluted twice to increase DNA yield)

• Extr. N-CPP
• Extr. Dig SOD E+A

DNA purification of "Dig. pMA.His (E+N)"

From 27/8
DNA clean-up using the E.Z.N.A. Gel Extraction kit, following procedures for DNA purification.

Elution volume: 30 μl (eluted twice to increase DNA yield)

• Pur. Dig. pMA His E+N

Gel verification

Since no DNA content was measurable for "Extr. Dig SOD E+A", a gel was run to verify DNA content in the three purified samples.

1 % agarose, 110 V

Samples:

• SOD: Extr. Dig SOD E+A
• pMA: Pur. Dig pMA.His E+N
• CPP: Extr. N-CPP cluster

Expected bands:

• SOD: 492 bp
• pMA: 2427 bp
• CPP: 379 bp

Results

• CPP resulted in a clear band of correct size.
• A very weak band at ≈500 bp is visible for SOD, which might be traces of DNA.
• No band visible for pMA.

Nevertheless, proceeded to ligation and cloning, hoping that the DNA is there.

Cloning of SOD into pMA.His

Ligation

	[μl]
Extr. Dig SOD E+A	12
Pur. Dig pMA.His E+N	3
5X Rapid Ligation buffer	4
T4 DNA ligase	1

Incubation: 22 °C, 10 min

Transformation

• 3 μl ligation mix. 30 min on ice.
• 30 sec heat shock in 42 °C
• Cells grown on Amp 100 LB agar, 37 °C

Amplification of N-CPPs from N-CPP cluster

Ran PCRs from the gel extracted N-CPP cluster with the following primers:

• Tra10: pSB-VF2 & pSB-VR
• TAT: pEXf & pEXr
• LMWP: pGexf & pGexr

Also ran 4 colony PCRs for Mimmi from cells transformed with pSB1C3.MITF plasmids (MITF 1-4); positive control (PC) pSB1C3.RFP; negative control (NC) blank; primers pSB-VF2 & pSB-VR.

PCR tubes

• illustra Ready-to-Go PCR beads
• 22.5 μl dH₂O
• 1 μl forward primer
• 1 μl reverse primer
• 0.5 μl template DNA

Standard colony PCR settings; 1:45 elongation time.

Gel verification

1 % agarose, 100 V

Expected bands:

• MITF: 1586 bp
• PC: 1385 bp
• Tra10: 182 bp
• TAT: 91 bp
• LMWP: 94 bp

Results

No relevant bands for MITF samples. Ran the gel too far for the CPP bands. Tra10 is still visible and seems correct. There might also be two bands at the very edge of the gel, supporting the 91 bp and 94 bp for TAT and LMWP, respectively.
Decided to go on with gel extraction of CPPs anyway.

Gel extraction

1 % agarose, 110 V. 22 μl sample (Tra10/TAT/LMWP)

Bands on UV table seemed correct relative to each other. Bands excised and gel excisions saved in -20 °C for later.

N-CPP

Plasmid prep

Prepared plasmid from Mimmi's 5 ml ON culture. 50 μl elution volume.

	[ng/μl]	A260/A280
N-CPP plasmid	671.9	1.94

Glycerol stock

Inoculated 3 ml LB + Amp 100 with 10 μl from Mimmi's ON culture. Incubated 6 h 37 °C, 225 rpm.
Prepared glycerol stock:

• N-CPP 28/8