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Lab notes (23/9 - 29/9)

Contents 1 Lab notes (23/9 - 29/9) 1.1 Futher PCR on POT2 with NinaB (New Primers NO. 5) 1.2 PCR on POT2 with NinaB (New Primers) 1.2.1 DNA purification from PCR 1.2.2 Restriction Digest 1.2.3 Gel purification 1.2.4 Ligation 1.2.5 Colony PCR on ligation from 24/8

Futher PCR on POT2 with NinaB (New Primers NO. 5)

Date: 23/8
Done by: Marie & Tommy
Methods: PCR
protocos:CP.1.1[1] Notes:
NinB2fw and NinaB2rv was used.
PCR were run programed as:

PCR	Temp. (C)	Time (min)
Start	95	2
Denaturing	95	1
Anneling	67,9	1
Elongation	72	4
End	72	5
Hold	4	indef.

PCR product from gradient PCR (d. 20/8-10), tube no. 5, was used as template.
The other tubes were pooled.

PCR on POT2 with NinaB (New Primers)

Start date: 24/8
Methods: Ligation, Competent cells, Transformation
Protocols: LG1.1[2], CC1.1[3], TR1.1[4]

DNA purification from PCR

Date: 20/8
Done by: Marie & Tommy
Methods: DNA purification from PCR
protocos:GFX purification from PCR - kit

One of the pooled tubes was eluted in 200µL, the others was eluted in 20µL
200µL nanodrop: 16,4 ng/µL
20µL nanodrop: 133,9ng/µL

Restriction Digest

Date: 24/8
Done by: Marie & Tommy
Methods: Restriction Digest
protocos:RD1.1[5] Notes:
Restriction mixture:

38 µL

8 µL

4 µL

4 µL

30 µL

Gel was run with uncut controles:

Gel purification

Date: 24/8
Done by: Marie & Tommy
Methods: gel purifikation
protocos:GFX gel purifikation kit Notes:
Purifide products was Nanodroped:
NinaB 1: 4,5 ng/µL
NinaB 2: 1,15 ng/µL
NinaB 3: 7,74 ng/µL
PSB1C3: 25,66 ng/µL
Nina B pooled: 4,5 ng/µL

Ligation

Date: 24/8
Done by: Marie & Tommy
Methods: Ligation
protocos:L1.3[6] Notes:
3 ligatons mixtures was made:
1:1 volumens 1 plasmid:5 insert 1:3 volumens 1 plasmid:15 insert 1:6 volumens 1 plasmid:30 insert

Colony PCR on ligation from 24/8

Date: 24/8
Done by: Marie & Tommy
Methods: Colony PCR
protocos:CP1.1[7] Notes:
15 colonies were picked form different plates, with differnt plasmid to insert ratio: colonie's 1,2,3,13 were picked from plates with 1:1 plasmid to insert ratio, colonie's 4,5,6 were picked from plates with 1:3 plasmid to insert ratio and colonie's 7,8,9,10,11,12,14,15 were picked from plates with 1:6 plasmid to insert ratio.
The PCR program was:

PCR	Temp. (C)	Time (min)
Start	95	2
Denaturing	95	1
Anneling	68,0	1
Elongation	72	4
End	72	5
Hold	4	indef.

A gel was run on the PCR products:

Futher experiments and PCR was run on tubes: 5,6,10,11 because they have the greatest yeild.