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Team:SDU-Denmark/labnotes6
From 2010.igem.org
(→Lab notes (16/9 - 22/9)) |
(→Lab notes (16/9 - 22/9)) |
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Done by: Marie & Tommy<br> | Done by: Marie & Tommy<br> | ||
Methods: Miniprep, Restriction digest, gel<br> | Methods: Miniprep, Restriction digest, gel<br> | ||
| - | Protocols: | + | Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2], [https://2010.igem.org/Team:SDU-Denmark/protocols#RD1.1 RD1.1]. |
Notes:<br> | Notes:<br> | ||
50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL. | 50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL. | ||
gel was run on produckt, showing recent miniprep compared to old miniprep produckt, showing uneven lengths. subsequent was performed.<br> | gel was run on produckt, showing recent miniprep compared to old miniprep produckt, showing uneven lengths. subsequent was performed.<br> | ||
| - | [[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-4.jpg | | + | [[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-4.jpg | 300px ]] |
==== Restriction digest on miniprep produckt (w. EcoRI) ==== | ==== Restriction digest on miniprep produckt (w. EcoRI) ==== | ||
Date: 17/8<br> | Date: 17/8<br> | ||
Done by: Marie & Tommy<br> | Done by: Marie & Tommy<br> | ||
Methods: Restriction digest, gel<br> | Methods: Restriction digest, gel<br> | ||
| - | Protocols: | + | Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#MP1.2 MP1.2], gel. |
Notes:<br> | Notes:<br> | ||
Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used<br> | Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used<br> | ||
The digest mix was incubated for 10 min at 37 C. A gel was run showing uncut new, cut new, uncut old and cut old miniprep product.<br> | The digest mix was incubated for 10 min at 37 C. A gel was run showing uncut new, cut new, uncut old and cut old miniprep product.<br> | ||
| - | [[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-5.jpg | | + | [[Image:Team-sdu-denmark-Minipreb_product_of_POT2_with_NinaB_1,5_gel_red_loader__-5.jpg |300px ]] |
=== PCR on POT2 with NinaB (New Primers) === | === PCR on POT2 with NinaB (New Primers) === | ||
Start date: 20/8<br> | Start date: 20/8<br> | ||
Methods: PCR, Gel<br> | Methods: PCR, Gel<br> | ||
| - | Protocols: | + | Protocols: [https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP.1.1] |
==== PCR on POT2 with NinaB (New Primers) ==== | ==== PCR on POT2 with NinaB (New Primers) ==== | ||
| Line 44: | Line 44: | ||
Done by: Marie & Tommy<br> | Done by: Marie & Tommy<br> | ||
Methods: ON<br> | Methods: ON<br> | ||
| - | protocos: | + | protocos:[https://2010.igem.org/Team:SDU-Denmark/protocols#CP1.1 CP1.1] |
Notes:<br> | Notes:<br> | ||
The new primers only contain the innermost restriction sites.<br> | The new primers only contain the innermost restriction sites.<br> | ||
| Line 56: | Line 56: | ||
</head> | </head> | ||
<body> | <body> | ||
| - | <table style="text-align: left; width: | + | <table style="text-align: left; width: 300px;" border="1" |
cellpadding="2" cellspacing="2"> | cellpadding="2" cellspacing="2"> | ||
<tr> | <tr> | ||
| Line 106: | Line 106: | ||
</head> | </head> | ||
<body> | <body> | ||
| - | <table style="text-align: left; width: | + | <table style="text-align: left; width: 300px;" border="1" |
cellpadding="2" cellspacing="2"> | cellpadding="2" cellspacing="2"> | ||
<tr> | <tr> | ||
Latest revision as of 22:21, 23 October 2010
Lab notes (16/9 - 22/9)
Contents |
Group: Retinal
ON of Top 10 E. coli cells with POT2 with NinaB
Date: 16/8
Done by: Marie & Tommy
Methods:
protocos:
Notes:
100 mL ON culture was made, cells were grown at 37 C in LB media with Chloramphenicol.
ON of Top 10 E. coli cells with POT2 with NinaB
Date: 17/8
Done by: Marie & Tommy
Methods: Miniprep, Restriction digest, gel
Protocols: MP1.2, RD1.1.
Notes:
50 mL of elution buffer was used. DNA concentrations after pooling were measured on NanoDrop to 206,2 ng/microL.
gel was run on produckt, showing recent miniprep compared to old miniprep produckt, showing uneven lengths. subsequent was performed.
Restriction digest on miniprep produckt (w. EcoRI)
Date: 17/8
Done by: Marie & Tommy
Methods: Restriction digest, gel
Protocols: MP1.2, gel.
Notes:
Due to the lack of old sample, restriction digest was performed using only 3,5 microL of miniprep produckt. 1,5 microL of H2O was added insted. EcoRI was used
The digest mix was incubated for 10 min at 37 C. A gel was run showing uncut new, cut new, uncut old and cut old miniprep product.
PCR on POT2 with NinaB (New Primers)
Start date: 20/8
Methods: PCR, Gel
Protocols: CP.1.1
PCR on POT2 with NinaB (New Primers)
Date: 20/8
Done by: Marie & Tommy
Methods: ON
protocos:CP1.1
Notes:
The new primers only contain the innermost restriction sites.
Meltting temp.: FWD: 68,1 C REV: 65,6 C
To get the optimal PCR temperatures a gradient PCR were run programed as:
| PCR | Temp. (C) | Time (min) |
| Start | 95 | 2 |
| Denaturing | 95 | 1 |
| Anneling | Gradient | 1 |
| Elongation | 72 | 4 |
| End | 72 | 5 |
| Hold | 4 | indef. |
The temperatur gradient were run from 60 to 70 C and the samples were run at these temperatures:
| Sample | Colunm | Temp. C |
| 1 | 1 | 59,9 |
| 2 | 3 | 60,7 |
| 3 | 5 | 62,7 |
| 4 | 7 | 65,4 |
| 5 | 9 | 67,9 |
| 6 | 11 | 69,6 |
After the PRC the product were run on a gel
The gel picture shows that alle of the temperatures gave results.
