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Lab notes (23/8 - 29/8)

Contents 1 Lab notes (23/8 - 29/8) 1.1 Photosensor 1.1.1 Insertion of B0015 in pSB3C5 and pSB3T5 1.1.1.1 Pfu PCR amplification and purification of B0015 1.1.1.2 Restriction digest of B0015 1.1.1.3 Ligation of PS and pSB1C3 and pCB1AK3 1.1.1.4 Transfomation of ligated plasmid in Top 10 E.coli 1.1.2 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

Insertion of B0015 in pSB3C5 and pSB3T5

Date: 8/23 - 8/29 2010
Done By: Maria and Lc
Protocol: CP1.1DE1.3RD1.1LG1.2CC1.1TR1.1CP1.3

Pfu PCR amplification and purification of B0015

Date: 8/23 2010
Done By: Maria and Lc
Protocol:CP1.1
Notes:
4 PCR reactions are prepared. 2uL Miniprep of pSB1AK3 (white 43) are distrubuted in each tube. PCR tubes are marked B00153.A-D.
Premix x5:

pfu buffer + MgSO4	25uL
dNTP mix	7.5uL
VF2 primer	7.5uL
VR primer	7.5uL
H20	190uL
pfu polymerase	2uL

PCR program:

start	94C	3min
denaturating	94C	2min
annealing	55C	30s
elongation	72C	30s
go to	2	rep.29x
end	72C	5min
hold	4C

5uL of the PCR product was loaded onto a 2% agarose gel. Gene ruler 100bp DNA ladder was used ad marker.

Results:
Analysis:
A strong band was observed at app. 450bp, and the rest of the PCR product was purified according to protocol using the GFX purification kit.
End conc.: 48ng/uL

Restriction digest of B0015

Date: 8/24 2010
Done By: Maria and Lc
Protocol:RD1.1DE1.3
Notes:
Restriction mixture B0015:

H2O	38uL
FD green buffer	8uL
EcoRI	4uL
PstI	4uL
B0015	30uL

The digested sample was loaded onto a 2% agarose extraction gel. Uncut B0015 was used as controle. Gene ruler 100bp DNA ladder was used as marker.
DNA was extracted from gel according to protocol.

Results:
DNA conc:

sample	conc. (ng/uL)
B0015	6.5
pSB3C5	50.8
pSB3T5	9.9

Analysis: the purified DNA was used for ligation.

Ligation of PS and pSB1C3 and pCB1AK3

Date: 8/24 2010
Done By: Maria and Lc
Protocol:LG1.2
Notes:
For each of the ligations three ligation mixtures were prepared. vector concentrations of 10n0g/uL (pSB3C5) and 50ng/uL (pSB3T5) respectively was used for each mixture. Appropiate amount of insert was added to reach vector:insert ratios of 1:1, 1:3 and 1:6 respectively.
Ligation mixtures (B0015 in pSB3C5):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB3C5	2uL	2uL	2uL
B0015	0.7uL	2uL	4.5uL
H20	14.3uL	13uL	10.5uL

Ligation mixtures (B0015 in pSB3T5):

	L1	L2	L3
T4 ligase buffer	2uL	2uL	2uL
T4 ligase	1uL	1uL	1uL
pSB3T5	5uL	5uL	5uL
B0015	0.5uL	1uL	2uL
H20	11.5uL	11uL	10uL

The samples was incubated at 17C ON at used for transformation

Transfomation of ligated plasmid in Top 10 E.coli

Date: 8/25 2010
Done By: Maria and Lc
Protocol:CC1.1TR1.1
Notes:
The compotent cells and transformation was carried out according to protocol.

Results:
the controle plates were okay, and there were many colonies on plates with cells transformed with either of the ligation mixtures..

Analysis:
The transformation was successfull and colonies was selected and used in coloni PCR.
--Tipi 13:41, 26 September 2010 (UTC)

Colony PCR on B0017

Date: 27/8
Done by: LC
Methods: PCR
Protocols: CP1.3
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:

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:CP1.1 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, CC1.1, TR1.1

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[1] 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 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 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 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 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.