"
Team:TU Delft/5 August 2010 content
From 2010.igem.org
(→Alkane degradation) |
|||
| Line 141: | Line 141: | ||
==Alkane degradation== | ==Alkane degradation== | ||
| - | === | + | ===Colony PCR=== |
| - | The morning started with a gel checking the PCR products of the one BioBrick that didn't give positives [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=5_August_2010 yesterday]. This gel showed one positive, but when I centrifuged the cells, they gave a red pellet. This indicated that the plasmid unfortunately was not the desired BioBrick, but the original destination plasmid. | + | The morning started with a gel checking the PCR products of the one BioBrick that didn't give positives [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=5_August_2010 yesterday]. This gel showed one positive, but when I centrifuged the cells, they gave a red pellet. This indicated that the plasmid unfortunately was not the desired BioBrick, but the original destination plasmid, so we'll have to try again. |
[[Image:TUDelft_20100805_PCR_020.png|350px|thumb|left|PCR of 020 colonies]] | [[Image:TUDelft_20100805_PCR_020.png|350px|thumb|left|PCR of 020 colonies]] | ||
| Line 171: | Line 171: | ||
===Plasmid Isolation=== | ===Plasmid Isolation=== | ||
| - | Of the colonies which gave a positive PCR, the plasmids were isolated using a | + | Of the colonies which gave a positive PCR [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=4_August_2010 yesterday], the plasmids were isolated using a QIA-gen miniprep kit. The following concentrations were obtained: |
| - | + | ||
| - | The following | + | |
{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | {| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" | ||
| Line 209: | Line 207: | ||
|} | |} | ||
| - | For some reason the 021K plasmid simply didn't want to be purified. All three of the isolates had very low concentration. | + | For some reason the 021K plasmid simply didn't want to be purified. All three of the isolates had a very low concentration. Next week we'll try another plasmid isolation method and see if that works. |
===Digestion=== | ===Digestion=== | ||
| + | |||
| + | |||
===Ligation=== | ===Ligation=== | ||
===Transformations=== | ===Transformations=== | ||
Revision as of 09:01, 6 August 2010
Contents |
Groningen Team
Today three members had an appointment with one of the professors at our department. We heard of this and invited them to come over to meet each other.
Lab Work
Alkane Sensing, Solvent Tolerance and Salt Tolerance
The plates containing yesterday's ligations contained colonies, to check whether they really contain the desired BioBrick a colony PCR was done, and the used colonies were grown in liquid LB medium over night. The results from the PCR were analysed on a 1% agarose gel.
Lane Description:
| # | Description | Expected Length (bp) | Primers | Status | Remarks |
| 1 | SmartLadder | n/a | n/a | n/a | |
| 2 | BBa_K398101 | 929 | G00101 + G00100 | ✓ | |
| 3 | BBa_K398101 | 929 | G00101 + G00100 | ✗ | |
| 4 | BBa_K398101 | 929 | G00101 + G00100 | ✗ | |
| 5 | BBa_K398101 | 929 | G00101 + G00100 | ✗ | |
| 6 | BBa_K398101 | 929 | G00101 + G00100 | ✗ | |
| 7 | BBa_K398402 | 758 | G00101 + G00100 | ✗ | |
| 8 | BBa_K398402 | 758 | G00101 + G00100 | ✗ | |
| 9 | BBa_K398402 | 758 | G00101 + G00100 | ✓ | |
| 10 | SmartLadder | n/a | n/a | n/a | |
| 11 | BBa_K398402 | 758 | G00101 + G00100 | ✗ | |
| 12 | BBa_K398402 | 758 | G00101 + G00100 | ✗ | |
| 13 | BBa_K398403 | 656 | G00101 + G00100 | ✗ | |
| 14 | BBa_K398403 | 656 | G00101 + G00100 | ✗ | |
| 15 | BBa_K398403 | 656 | G00101 + G00100 | ✗ | |
| 16 | BBa_K398403 | 656 | G00101 + G00100 | ✓ | |
| 17 | BBa_K398403 | 656 | G00101 + G00100 | ✗ |
Alkane degradation
Colony PCR
The morning started with a gel checking the PCR products of the one BioBrick that didn't give positives yesterday. This gel showed one positive, but when I centrifuged the cells, they gave a red pellet. This indicated that the plasmid unfortunately was not the desired BioBrick, but the original destination plasmid, so we'll have to try again.
Lane description
| # | Description | Primers | Expected length (bp) | ✓ | ✗ |
| L | SmartLadder | n/a | n/a | n/a | n/a |
| 1-9 | 020K | G00100 + G00101 | 2428 | 6 | rest |
Plasmid Isolation
Of the colonies which gave a positive PCR yesterday, the plasmids were isolated using a QIA-gen miniprep kit. The following concentrations were obtained:
| BioBrick | Composed of | Concentration (ng/μL) |
| 007A (1) | J61100-alkB2 | 33.8 |
| 007A (2) | J61100-alkB2 | 32.0 |
| 012K | J61100-rubR-B0015 | 42.7 |
| 021A | J61107-ALDH-B0015 | 46.3 |
| 021K (5) | J61107-ALDH-B0015 | 7.3 |
| 021K (1) | J61107-ALDH-B0015 | 11.1 |
| 021K (2) | J61107-ALDH-B0015 | 12.9 |
For some reason the 021K plasmid simply didn't want to be purified. All three of the isolates had a very low concentration. Next week we'll try another plasmid isolation method and see if that works.
