Spread DNA molecules

DNA spreading

Material

Lysis bufer
(200mM TrisHCl ph 7,4, 50mM EDTA, 0,5% SDS)

1mL
675 uL H2O
200 uL Tris.HCl pH 7.5
100 uL EDTA 500 mM
25 uL SDS 20%

Blocking solution
(2% BSA, 0,1% Tween 20, 1xPBS, 0,22 μL filtered), I add 0.02% NaN3 and reuse it, it can be filtered again.

70 mL	140mL
68.6 mL PBS 1x	137.2 mL PBS
1.4g BSA	2.8g BSA
70 uL TWEEN 20	140 uL TWEEN 20

Antibodies

Primary antibodies

Anti-BrdU/Idu (mouse), Becton Dickinson, Cat.No 347580
Anti-BrdU/CldU (rat), Abcam, Cat.No. ab6326.

Secondary antibodies

Anti-mouse Alexa 488, Invitrogen, Cat.No. A-11001
Anti-rat Cy3, Immuno Research, Cat.No. 712-166-1530
OR
ALEXA 555, goat anti-rat

Labelling

1. Seed cells on 6WP to be at ̴̴70% confluence – before additing drug. Prepare one special plate, which will be unlabelled cells.
2. Add 3μL CldU stock (5mg/mL= 19mM dilluted in dH2O) to 1 mL medium. Washing with PBS is not needed. 1mL is for 6WP.
3. Incubate 30 minutes (or as needed) at 37°C, 6% CO2.
4. Wash 3x with warm PBS.
5. Add 1mL fresh medium + 12 μl IdU stock (10mg/ml = 28,24 mM in DMSO or water + a few drops of 10M NaOH, until it dissolves), addition of IdU should not change the color of media.
6. Incubate 30 minutes (or as needed) at 37°C, 6% CO2.
7. Wash 2x with warm PBS.
8. Trypsinize labelled and unlabelled cells with 400 μl trypsin.
9. Add 1mL of DMEM+FBS, transfer to epp tube, centrifuge for 5 min, 1200 RPM, 4°C, wash with PBS once.
10. Resuspend in cold 1xPBS to 2.5*105 cells/mL. (count the cells)
11. Mix labelled cells 1:1 or 2 with unlabelled cells (depends on cell type and experiment)
12. Wipe glass slides with dH2O prior to mixing cells and lysis buffer, label it with pencil.
13. Mix 7,5 μl of lysis buffer (prepare shortly before) with 2,5 μl of cells directly on the slides. Firstly put the cells, then lysis buffer. Mix by gently stirring with the tip, then pipet 3x with P20 set to 5 μl. Be careful to keep the drop as small as possible. (After addition of SDS – be careful and do not make many bubbles)
14. Leave the slide horizontally for exactly 4.5 min at RT without moving. (Setup timer for every slide!)
15. Tilt the slide manually (30°-45°) to allow the drop to run slowly down the slide (Takes around 1 3 min to reach the bottom of the slide).
16. Air dry the slides completely (15-20 minutes) and fix with fresh solution of methanol/acetic acid 3:1 for 20 min RT. Then let dry overnight.

Immunolabelling:

1. Rehydrate slides in 1xPBS (2x3min).
2. Denature DNA in 2,5M HCl for 1h at RT. (Cover with aluminium foil) 32% HCl → 25ml/100ml; 37% HCl → 20ml/100ml, 35% HCl → 22ml/100ml
3. Wash several times in 1xPBS (5x3min) until pH is back to 7-7.5, not necessary to measure.

4. Block in block solution (freshly made and filtered - 0,22 μm ) for 20 min. – Take aliquote for antibodies. Don´t throw away the blocking solution after use, it is needed again before incubation in the 2. antibody. Cover it.

5. Incubate with primary antibody for 2 h at RT. Use 65 μl/slide.

• Drain slides briefly by canthing them on a tissue.
• Dilute the antibodies in blocking solution (both in the same tube).
• Rat anti-CldU – 1:500
• Mouse anti-IdU-FITC 1:100
• pipet 65 μl of antibody solution close to right side. Approach the slide and let the drop spread acroess the edge of it. Let down the slide carefully (the antibody solution should spread under the whole area of the slide). Cover the plastic tray with a lid and incubate in the dark.
  1. Wash in PBST 0,2% (5x3min)
  2. Dip 3x in blocking solution and drain slides briefly by canthing them on a tissue (Just leave it in the blocking solution while preparing the 2th Ab).
  3. Incubate with secondary antibody for 1 h at RT in the dark. Use 65 μl/slide.
• Dilute the antibody in blocking solution (both in the same tube)
• Anti-rat Cy3 1:150/Alexa anti rat 555 1:150
• Anti.mouse Alexa 488 1:300
  1. Wash in PBST 0,2% (5x3 min) – Then with 1xPBS (2x), dip the slide briefly in water
  2. Let slides air dry completely – 15-20 min.
  3. Mount coverslip with 20-30 μl/slide Antifade Gold or Fluoromont.
• Pipet the antifade gold evenly across one long side of the coverslip
• Lower a cover slip on the slide and let the Antifade gold spread across the whole slide.

Acquistion

Leica DM6000, 63x oil objective
200 molecules → 15-20 pictures on the slide (in the first half of the slide), 40 pictures are enough. If you need to quantify sister fork asymmetry, you can take more → optimal 7 fibers from the one picture