Chlamydomonas Phenol Chloroform DNA extraction_13697076

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title: "TheNessLab : Chlamydomonas Phenol-Chloroform DNA extraction"

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TheNessLab : Chlamydomonas Phenol-Chloroform DNA extraction

Created by Robert Ness, last modified on May 13, 2019

Adapted from: Genomic DNA Extraction (added by Leonardo Magneschi) 

1. Grow lawn of Chlamydomonas on 5-10cm Bold’s agar plate (500uL of liquid culture spread onto plate and grown for 3-5 days)

   1. Alternatively grow in liquid and pellet

2. Scrape lawn into 2ml round bottom tube and freeze at -80°C overnight. Final pellet needs to be quite large but the extraction is not very sensitive to the pellet size. Ranges from 0.1-0.3g of cells. ie a lot.

   1.  If grown in liquid, concentrate pellet and transfer to 2ml round bottom tube

3. Add 750 μl of 1:1 SDS-EB*: ddH20 buffer and glass bead to frozen sample.

4. Shake in homogenizer for 30sec at full strength

5. Spin tube down to compact the foam and create space for the following step

6. Add 750 μl phenol:chloroform:isoamyl alcohol (25:24:1), vortex

7. centrifuge 5 minutes at 13K x g (RCF) to separate phases

8. Transfer  aqueous phase (top clear layer) to a new tube. Careful not to transfer other layers

9. Add 2μl of RNase A and incubate 30-60 minutes at 37°C

10. Repeat step 6 - 8

11. Add equal volume chloroform, vortex

12. Centrifuge 5 minutes at 13K x g

13. Transfer aqueous phase to a new tube 1.5ml microcentrifuge tube

14. Add two volumes of 100% ethanol and incubate 1 hour at -20°C

    1. 1. Can be left in -20°C overnight

15. Centrifuge 20 minutes at 13K x g in 4^C centrifugre

16. Drain ethanol and wash the pellet once with 750μl 70% ethanol

17. Spin again for 1min

18. Drain 70% ethanol and spin again for 1min

19. Pipette off remaining ethanol

20. Dry pellet in flow hood for 5-10min and resuspend in 30-100μL ultrapure water or TE, depending on desired concentration

Buffers

**SDS-EB buffer:  **
2% SDS, 400 mM NaCl, 40 mM EDTA, 100 mM Tris-HCl (pH=8)

To make 1 litre:
1.  2% SDS:
% is grams/100ml therefore
20g SDS/litre (2g/100ml)

2. 400mM NaCl (MW = 58.44)
= .4 mols in a litre
= 0.4 * 58.44g/mol
= 23.376g (2.3376g/100ml)

3. 40mM EDTA (MW = 292.24)
= 0.04*292.24
= 11.6896g (1.16896g/100ml)

4. 100mM Tris-HCl (MW = 157.6)
= 0.1mols * 157.6g/mol
= 15.76g (1.576g/100ml)

Chemicals required

• SDS
• NaCl
• Tris-HCl
• EDTA
• Phenol:chloroform:isoamyl alcholo (25:24:1) pH 8.05 (http://products.invitrogen.com/ivgn/ product/15593031)
• Chloroform
• RNase A - Fermentas Thermo Scientific 10mg/ml recommended conc. 1-100μg/ml
• 100% EtOH
• 70% EtOH

Document generated by Confluence on May 22, 2024 11:44

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