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Team:SDU-Denmark/labnotes7
From 2010.igem.org
Lab notes (23/8 - 29/8)
Contents |
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.
Restriction Digest
Date: 24/8
Done by: Marie & Tommy
Methods: Restriction Digest
protocos:RD1.1[8]
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[9]
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. |
