Rui:LabNotes/SingleCell/2011-1-25: Difference between revisions
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==PCR result== | ==PCR result== | ||
Continued with 1.24.11 lab note [http://genome-tech.ucsd.edu/LabNotes/index.php?title=Rui:LabNotes/SingleCell/2011-1-24&action=edit§ion=5] | |||
===QPCR curve=== | ===QPCR curve=== | ||
1. Empty lane w/o tube | 1. Empty lane w/o tube |
Revision as of 19:29, 24 February 2011
PCR result
Continued with 1.24.11 lab note [1]
QPCR curve
1. Empty lane w/o tube
2. ES-500pg (4); EB-500pg (4)
3. ES-100pg (4); EB-100pg (4)
4. ES-10pg (4); EB-10pg (4)
5. NTC (4); NTC (4)
TBE PAGE gel
*4ul PCR product + 2ul loading dye *Low Mass ladder *6% TBE-PAGE gel, running at 200V for 30min *Stain w/ SYBR Gold for 3 min
M, NTC (1,2,5,6,7), M, 10pg (1,2,5,6,7)
100pg (1,2,3,5,8), M, 500pg (1,2,5,6,7), M
** Unspecific amplification from NTC, size range from 150-600bp ** Size shift from NTC to 500pg (600-2kb) was observed
PCR purification
- Combine 4 tubes for each sample
- Add 16ul NaOAc and 400ul EtOH (R.T.), mix
- Incubate -80C for 1 hour
- Centrifuge at 10,000rpm 4C 30min: observed a huge white pellet in each tube; probably Qiagen PCR kit or beads should be used
- Wash with 70% EtOH (R.T.)
- Resolve in 36ul Nf water, store at -80C
- TA cloning [2]