Matt:LabNotes/2013-7-1: Difference between revisions

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>Mzcai
(Created page with "====Amplification with Sequencing Adapters==== *Common linker sequence in every probe is: CTTCAGCTTCCCGATATCCGACGGTAGTGT (Porecca et al. 2007) **[[Media:probe2padlockFISSEQ_2...")
 
>Mzcai
 
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-28
====Amplification with Sequencing Adapters====
====Amplification with Sequencing Adapters====


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*Since this is the same design as LC Sciences probes that Noi used, I'll use the same [http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Primers_for_LC_Sciences_library-free_protocol primers]
*Since this is the same design as LC Sciences probes that Noi used, I'll use the same [http://genome-tech.ucsd.edu/LabNotes/index.php/Illumina_GA/Oligo_info#Primers_for_LC_Sciences_library-free_protocol primers]
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Tube #'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Index#'''
|-
| 1||NTC-0gap||Indx7
|-
| 2||gDNA-0gap||Indx45
|-
| 3||cDNA-0gap||Indx76
|-
| 4||NTC-20gap||Indx7
|-
| 5||gDNA-20gap||Indx77
|-
| 6||cDNA-20gap||Indx78
|-
|
|} <br>Will also do duplicate for each sample so 12 reactions total<br>
{| {{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1 rxn'''
| align="center" style="background:#f0f0f0;"|'''13 rxn'''
|-
| Captured template||12.00||0.00
|-
| 10uM Forward+Indx||2.00||0.00
|-
| 10uM Reverse||2.00||26.00
|-
| 2x KAPA SYBG fast MM||50.00||650.00
|-
| H2O||34.00||442.00
|-
| Total||100.00||1300.00
|}


   Program
   Program
   *98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x7 cycles -> 72°C 3 min -> 15°C hold
   *98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x7 cycles -> 72°C 3 min -> 15°C hold
[[File:07012013_Agi26kCapturedSequencePCRGraph.JPG | 650px]]
*Sample with greatest amplification is NTC for 20gap probes
**Will run a gel to see what is causing the signal
*Both 20gap samples did not show amplification suggesting something went wrong
**Possibly the dNTP mix used during capture reaction is to blame because Noi says the Stoffel fragment enzyme was working a week ago
**Will do a PCR with the same dNTP mix to see
*Both 0gap samples showed moderate amplification and NTC was as expected
PCR'd an additional 3 cycles to make a total of 21 to match CustomArray
  Program
  *98°C 30sec -> (98°C 10sec -> 72°C 2min30sec) x3 cycles
====Gel Check====
[[File:2013-07-01_Agi26kCapturedPCR_GelCheck.jpg | 450px]]
*Looks like I mixed up 20gap samples while pipetting but I'm not sure how
**I was very careful about adding correct captured template to each PCR reaction
**If this was caused by switching two samples, then expect to see two bright bands and one dim/nonexistent one
**Also each sample is consistent with its duplicate in qPCR curve, and it is unlikely I would pipette incorrectly twice
**Also a faint band around 300bp can still be seen in samples (possibly will be brighter after 3 more amplification cycles)
*It is possible a mistake happened during capture reaction but I was also very careful with putting reagents in the right tubes during that step
*Another possibility is that NTC-20gap had contamination somehow and everything else works fine
**Will know more after testing dNTP mix tomorrow
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-2

Latest revision as of 17:02, 2 July 2013

Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-6-28

Amplification with Sequencing Adapters[edit]

  • Since this is the same design as LC Sciences probes that Noi used, I'll use the same primers
Tube # Sample Index#
1 NTC-0gap Indx7
2 gDNA-0gap Indx45
3 cDNA-0gap Indx76
4 NTC-20gap Indx7
5 gDNA-20gap Indx77
6 cDNA-20gap Indx78


Will also do duplicate for each sample so 12 reactions total

Components 1 rxn 13 rxn
Captured template 12.00 0.00
10uM Forward+Indx 2.00 0.00
10uM Reverse 2.00 26.00
2x KAPA SYBG fast MM 50.00 650.00
H2O 34.00 442.00
Total 100.00 1300.00
 Program
 *98°C 30sec -> (98°C 10sec -> 52°C 30sec -> 72°C 30sec) x8 cycles -> (98°C 10sec -> 72°C 30sec) x7 cycles -> 72°C 3 min -> 15°C hold

File:07012013 Agi26kCapturedSequencePCRGraph.JPG

  • Sample with greatest amplification is NTC for 20gap probes
    • Will run a gel to see what is causing the signal
  • Both 20gap samples did not show amplification suggesting something went wrong
    • Possibly the dNTP mix used during capture reaction is to blame because Noi says the Stoffel fragment enzyme was working a week ago
    • Will do a PCR with the same dNTP mix to see
  • Both 0gap samples showed moderate amplification and NTC was as expected

PCR'd an additional 3 cycles to make a total of 21 to match CustomArray

 Program
 *98°C 30sec -> (98°C 10sec -> 72°C 2min30sec) x3 cycles

Gel Check[edit]

File:2013-07-01 Agi26kCapturedPCR GelCheck.jpg

  • Looks like I mixed up 20gap samples while pipetting but I'm not sure how
    • I was very careful about adding correct captured template to each PCR reaction
    • If this was caused by switching two samples, then expect to see two bright bands and one dim/nonexistent one
    • Also each sample is consistent with its duplicate in qPCR curve, and it is unlikely I would pipette incorrectly twice
    • Also a faint band around 300bp can still be seen in samples (possibly will be brighter after 3 more amplification cycles)
  • It is possible a mistake happened during capture reaction but I was also very careful with putting reagents in the right tubes during that step
  • Another possibility is that NTC-20gap had contamination somehow and everything else works fine
    • Will know more after testing dNTP mix tomorrow

Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-7-2