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Team:SDU-Denmark/labnotes7
From 2010.igem.org
Lab notes (23/8 - 29/8)
Contents |
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[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:
| 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. |
==== 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. |
