Daniel:Notebook/HiResChrPaint/2013-7-24

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S2 Probe Prep (Started 7/23/2013)[edit]

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FISH Slide Preparation[edit]

1. Rinse slides in 100% ethanol; allow slides to dry completely 2. Prepare a cell suspension of 500 000-2 000 000 cells/mL in growth media 3. Add 100 uL of cell suspension per slide roughly in the center 4. Allow cells to adhere for 1-3 hours at the growth temp of the cell line 5. Rinse slides briefly in 1X PBS 6. Fix slides for 5-15 minutes in 4% (v/v) paraformaldehyde in 1X PBS 7. Rinse briefly in 1X PBS in a coplin jar 8. Incubate for 5 minutes in 2X SSCT 9. Incubate for 5 minutes in 2X SSCT + 50% (v/v) formamide 10. Transfer to fresh container of 2X SSCT + 50% (v/v) formamide for storage at 4C 11. Slides are typically best used within 1-2 weeks of creation

DpnII Digestion (From yesterday)[edit]

3. Pool samples for V4 set into 4AB and 4CD
4. Column Purification, elute with 30 uL EB
5. Measure concentrations in nanodrop

Nanodrop Results[edit]

Sample V4S2AB V4S2CD V6S2A V6S2B V6S2C V6S2D
ng/uL 114.5 112.8 101.1 105.6 99 109.4
ug in 30 uL 3.4 3.4 3.0 3.2 3.0 3.3
total ug   6.8 12.5

DpnII Digest gel[edit]

1. Lanes; (5.5 uL TBE; 2 uL 6x buffer, 0.5 uL sample; load 7 uL for all)


Lane 1 2 3 4 5 6 7 8 9 10 11 12
Sample 25bp ladder V4S2AB V4S2CD V6S2A V6S2B V6S2C V6S2D

Gel Image

File:DpnIIDigest 72413.jpg

Lambda Exonuclease Digestion[edit]

1. Reaction Table
Sample V4S2AB V4S2CD V6S2A V6S2B V6S2C V6S2D V4 (from 7/23/2013) V6 (from 7/23/2013)
ng/uL 114.5 112.8 101.1 105.6 99 109.4 142.9 ~33
ug in 25 uL 2.9 2.8 2.5 2.6 2.5 2.7 NA NA
uL added 25 25 25 25 25 25 18 32
uL 10x buffer 5 5 5 5 5 5 5 5
uL Lambda Exonuclease (5U/uL) 3 3 3 3 3 3 3 3
uL nfH2O 17 17 17 17 17 17 24 10
total 50 50 50 50 50 50 50 50
2. Incubate for 1 hour at 37C followed by 10 minute heat inactivation at 75C
3. Pool samples
4. ssDNA column purification, elute with 20 uL
5. Measure Concentration in Nanodrop
6. Dry samples in vaccuum centrifuge

Nanodrop Results[edit]

  ng/uL ug in 20 uL
V4S2 274.1 5.5
V6S2 193 3.9