Rui:LabNotes/SingleCell/2011-1-25: Difference between revisions
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>Zsakura2 |
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==PCR purification== | ==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 | **observed a huge white pellet in each tube | ||
**Qiagen PCR purification kit should be used as mentioned in original protocol | **Qiagen PCR purification kit should be used as mentioned in original protocol | ||
#Wash with 70% EtOH (R.T.) | |||
#Resolve in 36ul Nf water, store at -80C |
Revision as of 19:20, 24 February 2011
PCR result
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 **Qiagen PCR purification kit should be used as mentioned in original protocol #Wash with 70% EtOH (R.T.) #Resolve in 36ul Nf water, store at -80C