Matt:LabNotes/2016-5-25: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
mNo edit summary
>Mzcai
mNo edit summary
Line 84: Line 84:
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold


[[ | 650px]]
[[File:20160526_TB12k_ExpansionPCRtest.JPG | 450px]]
*1,000X final dilution was in the sweet spot for number of cycles (15-20)
*1,000X final dilution was in the sweet spot for number of cycles (15-20)
*Use that concentration and do 19 cycles
*Use that concentration and do 19 cycles
Line 103: Line 103:
| Total||150
| Total||150
|}
|}
 
*50ul per PCR tube
'''Program'''
'''Program'''
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold
95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold

Revision as of 00:26, 27 May 2016

TB12k_Apr2016 Probe Preparation

  • Probe Design
  • Received 195-mer oligos 5/25/26
    • Name: TB12k_Apr2016_DARTFISH
    • Amount: 341ng
    • Physical State: Solid
  • Calculate MW of 195 nt probe = 195nt * 303.7Da/nt + 79Da = 59,300.5 g/mol
  • 341ng -> 5.75pmol

Resuspend and Aliquot

  • Resuspend in 100ul (57.5nM)
    • Pipetted 20X to mix
  • Made aliquots of 40, 40, 10, and 10 into 0.5ml Lo-Bind tubes

Serial Dilution 1000X

  • Take 1ul from a 10ul aliquot and add 9ul H2O (10X Dilution)
  • Take 5ul and add 45ul H2O (100X Dilution)
  • Take 20ul and add 180ul H2O (1000X Dilution)

Expansion PCR Test

  • NTC
Component Volume
Seed oligo 0
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 10.5
Total 25
  • Positive Control
Component Volume
CA12kNov2014_V4 1st rnd Oligos (10nM) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 1,000X Dilution
Component Volume
Seed oligo (100X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25
  • 10,000X Dilution
Component Volume
Seed oligo (1000X Dilution) 2.5
F/R V4 Primer Mix (10uM) 2
2X Kapa MM 12.5
H2O 8
Total 25

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 40 -> 72C 2min -> 15C hold

File:20160526 TB12k ExpansionPCRtest.JPG

  • 1,000X final dilution was in the sweet spot for number of cycles (15-20)
  • Use that concentration and do 19 cycles

Expansion PCR

Component Volume
Seed oligo (100X Dilution) 15
F/R V4 Primer Mix (10uM) 12
2X Kapa MM 75
H2O 48
Total 150
  • 50ul per PCR tube

Program 95C 30sec -> (95C 30sec -> 55C 45sec-> 72C 45sec) x 19 -> 72C 2min -> 15C hold