"
09/08/13
From 2013.igem.org
(Difference between revisions)
AliceHaworth (Talk | contribs) |
AliceHaworth (Talk | contribs) (→Isolating plasmid) |
||
| Line 33: | Line 33: | ||
|} | |} | ||
| + | ==Digesting the plasmids for restriction mapping== | ||
| + | *Digesting 200ng of DNA with SacI | ||
| + | **1ul of SacI | ||
| + | **2ul of NEB buffer 1.1 | ||
| + | **DNA volumes added for samples 5.1 to 10.3: | ||
| + | ***3ul; 5ul; 4ul; 11.5ul | ||
| + | **Water added for samples 5.1 to 10.3: | ||
| + | ***14ul; 12ul; 13ul; 5.5ul | ||
| + | *Digestion in 37C water bath for 30mins | ||
| + | *Heat kill at 80C for 20mins | ||
| + | |||
| + | ==Running an agarose gel== | ||
| + | *Add 5ul of 6x orange G to each sample | ||
==Glycerol stocks== | ==Glycerol stocks== | ||
Revision as of 14:45, 9 August 2013
| Home | Team | Official Team Profile | Project | Parts Submitted to the Registry | Modeling | Notebook | Safety | Attributions |
|---|
Contents |
Isolating plasmid
- From overnight culture, took 3ml of culture and centrifuged into pellet of samples 5.1, 10.1, 10.2, 10.3
- Isolated the plasmid using omega bio-tek Plasmid mini kit I
- Concentrations from nano drop are shown in the table below
| Sample | Volume(ul) | Conc.(ng/ul) | 260/280 | 260/230 |
| 5.1 | 92 | 65.8 | 1.87 | 1.75 |
| 10.1 | 88 | 36.9 | 1.80 | 1.77 |
| 10.2 | 89 | 48.3 | 1.79 | 1.98 |
| 10.3 | 94 | 17.4 | 1.84 | 1.59 |
Digesting the plasmids for restriction mapping
- Digesting 200ng of DNA with SacI
- 1ul of SacI
- 2ul of NEB buffer 1.1
- DNA volumes added for samples 5.1 to 10.3:
- 3ul; 5ul; 4ul; 11.5ul
- Water added for samples 5.1 to 10.3:
- 14ul; 12ul; 13ul; 5.5ul
- Digestion in 37C water bath for 30mins
- Heat kill at 80C for 20mins
Running an agarose gel
- Add 5ul of 6x orange G to each sample
Glycerol stocks
- Took 750ul from overnight stock and centrifuged
- Supernatant was removed
- Pellet resuspended in 375ul of HMFM
- samples were then frozen at -80
Restriction mapping
- using isolated plasmid samples 5.1, 10.1, 10.2, 10.3
