927
edits
Project:Plant species testing: Difference between revisions
From London Hackspace Wiki
→Chelex extraction method - 17 and 18/7/12
Mycoplasma (talk | contribs) |
Mycoplasma (talk | contribs) |
||
| Line 29: | Line 29: | ||
*Extraction: same procedure, but started with approx 50 mg of pea in 600ul chelex. (This is much more reactant than in the paper and a waste of chelex, but our scale only goes down to 100mg. Would be good to get a smaller one.) | *Extraction: same procedure, but started with approx 50 mg of pea in 600ul chelex. (This is much more reactant than in the paper and a waste of chelex, but our scale only goes down to 100mg. Would be good to get a smaller one.) | ||
*Electrophoresis: same procedure but used 0.5% instead of 1% agarose solution as we are running genomic DNA not PCR product. | *Electrophoresis: same procedure but used 0.5% instead of 1% agarose solution as we are running genomic DNA not PCR product. | ||
Saw a faint, blurry band, running ahead of the ladder. Not sure what this is, as genomic DNA should be behind the ladder if not still in the well. | |||
==== Repeat without PCR: 30/7/12 ==== | ==== Repeat without PCR: 30/7/12 ==== | ||
* Bashed peas into paste in a glove | * Bashed peas into paste in a glove | ||
* Placed a sample of template in 250 ul of chelex 5% | * Placed a 1/2g of sample of template in 250 ul of chelex 5% | ||
* Incubated at 56°C for 30 mins, with periodic vortexing, the 94°C for 10 mins | * Incubated at 56°C for 30 mins, with periodic vortexing, the 94°C for 10 mins | ||
* | * 10 ul of supernatant visualised on 1% agarose gel, together with a sample containing only loading dye, a sample containing only chelex, and a sample from Will's first chelex extraction of 17/7. | ||
Conclusion: we think DNAses may be affecting the sample. | Chelex only and loading dye only samples had no bands - so they are not contaminated with DNA. All pea samples showed a band running ahead of the ladder, becoming more diffuse over time. Conclusion: we think DNAses may be affecting the sample, cutting the DNA into small pieces. | ||
== Precipitation extraction method: 17/7/12, 24/7/12 == | == Precipitation extraction method: 17/7/12, 24/7/12 == | ||
