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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
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
|