Rui:LabNotes/SingleCell/2011-1-25

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Revision as of 19:56, 24 February 2011 by >Zsakura2
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PCR result

Continued with 1.24.11 lab note [1]

QPCR curve

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

*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

  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

[3]