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:1.24.11-L1]]
[[File:1.24.11-L1]]
Line 15: Line 17:
[[File:1.24.11-L6]]
[[File:1.24.11-L6]]


==TBE PAGE gel==
===TBE PAGE gel===
  *4ul PCR product + 2ul loading dye
  *4ul PCR product + 2ul loading dye
  *Low Mass ladder
  *Low Mass ladder
Line 26: Line 28:
  100pg (1,2,3,5,8), M, 500pg (1,2,5,6,7), M
  100pg (1,2,3,5,8), M, 500pg (1,2,5,6,7), M
[[File:Example.jpg]]
[[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

File:1.24.11-L1

2. ES-500pg (4); EB-500pg (4)

File:1.24.11-L2

3. ES-100pg (4); EB-100pg (4)

File:1.24.11-L3

4. ES-10pg (4); EB-10pg (4)

File:1.24.11-L5

5. NTC (4); NTC (4)

File:1.24.11-L6

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)

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[edit]

  1. Combine 4 tubes for each sample
  2. Add 16ul NaOAc and 400ul EtOH (R.T.), mix
  3. Incubate -80C for 1 hour
  4. Centrifuge at 10,000rpm 4C 30min: observed a huge white pellet in each tube; probably Qiagen PCR kit or beads should be used
  5. Wash with 70% EtOH (R.T.)
  6. Resolve in 36ul Nf water, store at -80C
  7. TA cloning [2]

PCR protocol trouble shooting[edit]

[3]