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] | |||
===QPCR curve=== | |||
1. Empty lane w/o tube | 1. Empty lane w/o tube | ||
[[File: | [[File:1.24.11-L1]] | ||
2. ES-500pg (4); EB-500pg (4) | 2. ES-500pg (4); EB-500pg (4) | ||
[[File:1.24.11-L2]] | |||
3. ES-100pg (4); EB-100pg (4) | 3. ES-100pg (4); EB-100pg (4) | ||
[[File:1.24.11-L3]] | |||
4. ES-10pg (4); EB-10pg (4) | 4. ES-10pg (4); EB-10pg (4) | ||
[[File:1.24.11-L5]] | |||
5. NTC (4); NTC (4) | 5. NTC (4); NTC (4) | ||
[[File:1.24.11-L6]] | |||
==TBE PAGE gel== | ===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) | M, NTC (1,2,5,6,7), M, 10pg (1,2,5,6,7) | ||
[[File:2011-01-25-NTC-10.jpg]] | |||
100pg (1,2,3,5,8), M, 500pg (1,2,5,6,7), M | |||
[[File:Example.jpg]] | |||
** 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 [http://genome-tech.ucsd.edu/LabNotes/index.php?title=Rui:LabNotes/SingleCell/2011-1-26] | |||
==PCR protocol trouble shooting== | |||
[http://genome-tech.ucsd.edu/LabNotes/index.php?title=Rui:LabNotes/SingleCell/2011-1-26] |
Latest revision as of 19:57, 24 February 2011
PCR result[edit]
Continued with 1.24.11 lab note [1]
QPCR curve[edit]
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[edit]
*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[edit]
- 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]