"
Team:TU Delft/9 August 2010 content
From 2010.igem.org
(Difference between revisions)
(→Colony PCR) |
(→Alkane degradation) |
||
| Line 130: | Line 130: | ||
==Colony PCR== | ==Colony PCR== | ||
[https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=06_August_2010 Friday], a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length. | [https://2010.igem.org/Team:TU_Delft#page=pages/blog&blog=06_August_2010 Friday], a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length. | ||
| + | |||
| + | =Emulsifier= | ||
Revision as of 08:23, 10 August 2010
Contents |
Alkane degradation
Digestion, Ligation, Transformation
A digestion was done on a number of BioBricks for the production of new BioBricks.
| # | Sample | Enzyme 1 | Enzyme 2 | Enzyme 3 | Buffer | BSA | Needed fragment |
| 1 | 009A | EcoRI | SpeI | PvuI | 2 (Biolabs) | ✓ | ‘E - J61100 - rubA4 - S’ |
| 2 | 012K | EcoRI | XbaI | ✗ | 2 (Biolabs) | ✓ | ‘E – J61100 - rubR - B0015 – X’ |
| 3 | 017A | SpeI | PstI | 2 (Biolabs) | ✓ | ‘P - pSB1A2 – J61100 - ladA – S’ | |
| 4 | 018A | XbaI | PstI | PvuI | 3 (Biolabs) | ✓ | ‘X – J61107 - ALDH – P’ |
The digestion was checked on a gel, together with the uncut BioBricks:
Lane description
| # | Description | Expected size (bp) | OK? | # | Description | Expected size (bp) | OK? |
| L | Smartladder (3μl) | n/a | n/a | 5 | 017A cut | ||
| 1 | 009A cut | 6 | 017A uncut | n/a | |||
| 2 | 009A uncut | n/a | 7 | 018A cut | |||
| 3 | 012K cut | 8 | 018A uncut | n/a | |||
| 4 | 012K uncut | n/a |
Ligation
Following the digestions, the fragments were [Team:TU_Delft/protocols/ligation ligated]] for 4 hours.
| # | BioBrick | Fragment 1 | Recipient vector |
| 1 | 013AK | 10 μL ‘E – J61100 - rubA4 – S’ | 10 μL ‘X – J61100 - rubR - B0015 - pSB1AK3 – E’ |
| 2 | 020A | 10 μL ‘P - pSB1A2 – J61100 - ladA – S’ | 10 μL ‘X – J61107 - ALDH – P’ |
Colony PCR
Friday, a colony PCR on colonies from a plate with transformants with BioBrick 013AK did not give positive results. But this doesn't mean that none of the colonies are correct. To check we did another colony PCR today on 8 more colonies of the AMP plate as well as the KAN plate. Hopefully a the PCR product of a colony will show the correct length.
