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Lab notes (23/8 - 29/8)

Contents 1 Lab notes (23/8 - 29/8) 1.1 Photosensor 1.1.1 Colony PCR on B0017 1.2 Retinal 1.2.1 Futher PCR on POT2 with NinaB (New Primers NO. 5) 1.2.2 PCR on POT2 with NinaB (New Primers) 1.2.2.1 DNA purification from PCR 1.2.2.2 Restriction Digest 1.2.2.3 Gel purification 1.2.2.4 Ligation 1.2.2.5 Colony PCR on ligation from 24/8 1.2.2.6 Restriction Digest 1.2.2.7 Colony PCR on ligation from 24/8 (colonies 5,6,10 and 11 + new colonies)

Photosensor

Colony PCR on B0017

Date: 27/8
Done by: LC
Methods: PCR
Protocols: CP1.3[1]
Notes:
Premix:
12,5 µl 10xTAQ Buffer
5 µl MgCl2
5 µl VF2
5 µl VR
2,5 µl dNTP
17,5 µl H2O
5/8 µl TAQ Polymerase

9,5 µl Premix were added to 15 µl of H2O containing the lysed cells.

PCR Program:

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Start	94  C	2 min
Denaturing	94 C	1 min
Annealing	55 C	1 min
Elongation	72 C	30 sec
Goto2	rep	29x
End	72 C	3 min
Hold	4 C

Results: No useable results, only unclear bands around 120 BP, which seem to be the result of mispriming with VR on B0010.

Retinal

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

Date: 23/8
Done by: Marie & Tommy
Methods: PCR
protocos:CP.1.1[2] 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[3], CC1.1[4], TR1.1[5]

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[6] 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[7] 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[8] 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.

Restriction Digest

Date: 24/8
Done by: Marie & Tommy
Methods: Restriction Digest
protocos:RD1.1[9] Notes:
Restriction digest was performed on tubes 5,6,10 and 11 to test for insertion of ninaB (Correct orientation) XbaI and SPEI was used and a gel was run:

Colony PCR on ligation from 24/8 (colonies 5,6,10 and 11 + new colonies)

Date: 24/8
Done by: Marie & Tommy
Methods: Colony PCR
protocos:CP1.1[10] Notes:
8 new colonies (16-23) were chosen in addition to colonies 5,6,10 and 11 form 25I8-10 colonie PCR.
PCR were run with TAQ polymerase and VF2 + VR primers according to the following program:

PCR	Temp. (C)	Time (min)
Start	94	2
Denaturing	94	1
Anneling	55,0	0.5
Elongation	72	2
End	72	5
Hold	4	indef.

A gel was run on the products:
==== PCR on NinaB with old primers (NinaBfw and NinaBrv) ==== Date: 27/8
Done by: Marie & Tommy
Methods: PCR
protocos:CP1.1[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1] Notes:
PCR was run on NinaB (from PCR with NinaBfw and NinaBrv), this time with the old primers (NinaBfw and NinaBrv) in an attempt to add the E and P restriction sites to the NinaB fracment.
The PCR program was set to:

PCR	Temp. (C)	Time (min)
Start	95	2
Denaturing	95	1
Anneling	55,0	0.75
Elongation	72	2
Denaturing	94	1
Anneling	73,0	0.75
Elongation	72	2
End	72	5
Hold	4	indef.

NinaB From second new primer PCR (23/8, purifid 24/8) was used as templated.