Matt:LabNotes/2013-9-25: Difference between revisions
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5. Added 2ul of dye to each sample | 5. Added 2ul of dye to each sample | ||
6. Incubated in dark for 1 hr | 6. Incubated in dark for 1 hr | ||
7. Added 5ul | 7. Added 15ul H2O to dye-coupled samples to make 25ul total | ||
8. | 8. Took 0.5ul of dye-coupled sample and diluted to 10ul for '''non-filtered measurements''' | ||
9. Added 24.5ul of sample to prepped Centri-Sep column and spun for 2 min at 750rcf | |||
10. Measured dye conc and DNA conc using Nanodrop Microarray setting (dsDNA) | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Alexa 564 (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''DNA conc (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''DNA (pmol/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Bases (pmole/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Base:Dye Ratio''' | |||
|- | |||
| Filtered||+aadUTP||0.5||20.3||0.346074442||62.29339953||124.5867991 | |||
|- | |||
| Filtered||-aadUTP||0||25.5||0.434724053||78.25032945|| | |||
|- | |||
| Filtered||NTC||0||0.5||0.008524001||1.534320185|| | |||
|- | |||
| Nonfiltered||+aadUTP||17.7||14.7||0.25060563||45.10901345||2.548531833 | |||
|- | |||
| Nonfiltered||-aadUTP||16.3||13.9||0.236967229||42.65410115||2.616816022 | |||
|- | |||
| Nonfiltered||NTC||17.8||14.3||0.243786429||43.8815573||2.465256028 | |||
|} | |||
*90bp DNA mass: 58,658 g/mol | |||
*The Nonfiltered NTC is messed up, possibly could've taken the 0.5 ul from wrong tube in step 8 | |||
*Among the filtered samples, good evidence that the aadUTP is still good | |||
**Base:Dye ratio isn't the best but definitely falls within the range that Dan gets [[Daniel:ProbeList]] | |||
**He also uses 3x more dye for a reaction which could explain why I got less dye/base |
Latest revision as of 00:30, 26 September 2013
Expected number of ACTB and RAB7A Rolonies[edit]
- Initial trial of secondary gene rolony generation showed low number of rolonies http://genome-tech.ucsd.edu/LabNotes/index.php/Hosuk:LabNotes/2013-9-12#Number_of_Rolonies
- I want to look at literature to see how many rolonies of ACTB and RAB7A we should expect
- http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM313643 is gene expression data for PGP1F cells using Illumina Ref-8 v3.0 beadchip (targets 18,631 unique genes)
- The table at the end of http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL6883 can be used to convert gene symbol to ILMN ID Ref
' | ID Ref | ID Ref | Expression Value | Expression Value | % of total |
ACTB | ILMN_2152131 | ILMN_2038777 | 42,723.45 | 33,579.69 | 0.32 |
RAB7A | ILMN_1716524 | N/A | 7,254.71 | N/A | 0.030 |
All Transcripts | N/A | N/A | 23,976,360 | N/A | 100 |
- According to this: http://bfg.oxfordjournals.org/content/8/6/437.full.pdf, 95% of RNA is housekeeping ncRNA. In other words ~5% of our primary rolonies is mRNA
- Therefore expect 0.016% of primary rolonies to be ACTB secondary rolonies
- Expect 0.0015% of primary rolonies to be RAB7A secondary rolonies
Dye-coupling aminoallyl-dUTP[edit]
Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-9-24
- Following Dan's Dye Coupling Protocol and Dan's Column Size Separation Protocol
- 3 5ul samples: +aadUTP, -aadUTP, and NTC (just water)
1. Warm DMSO and sodium bicarbonate buffer to RT 2. Added 3ul of of sodium bicarbonate to 5ul sample 3. Denatured samples at 95C for 5 min and then snap cooled 4. Dissolved 1 vial of Alexa 564 dye in 6ul DMSO; vortexed and spun down 5. Added 2ul of dye to each sample 6. Incubated in dark for 1 hr 7. Added 15ul H2O to dye-coupled samples to make 25ul total 8. Took 0.5ul of dye-coupled sample and diluted to 10ul for non-filtered measurements 9. Added 24.5ul of sample to prepped Centri-Sep column and spun for 2 min at 750rcf 10. Measured dye conc and DNA conc using Nanodrop Microarray setting (dsDNA)
' | ' | Alexa 564 (pmol/ul) | DNA conc (ng/ul) | DNA (pmol/ul) | Bases (pmole/ul) | Base:Dye Ratio |
Filtered | +aadUTP | 0.5 | 20.3 | 0.346074442 | 62.29339953 | 124.5867991 |
Filtered | -aadUTP | 0 | 25.5 | 0.434724053 | 78.25032945 | |
Filtered | NTC | 0 | 0.5 | 0.008524001 | 1.534320185 | |
Nonfiltered | +aadUTP | 17.7 | 14.7 | 0.25060563 | 45.10901345 | 2.548531833 |
Nonfiltered | -aadUTP | 16.3 | 13.9 | 0.236967229 | 42.65410115 | 2.616816022 |
Nonfiltered | NTC | 17.8 | 14.3 | 0.243786429 | 43.8815573 | 2.465256028 |
- 90bp DNA mass: 58,658 g/mol
- The Nonfiltered NTC is messed up, possibly could've taken the 0.5 ul from wrong tube in step 8
- Among the filtered samples, good evidence that the aadUTP is still good
- Base:Dye ratio isn't the best but definitely falls within the range that Dan gets Daniel:ProbeList
- He also uses 3x more dye for a reaction which could explain why I got less dye/base