Hello,

Although a lot of people use liquid nitrogen to freeze the cell and grind the cell wall fo the frozen cell using mortar and pestle, I prefer using glass beads and a Fastprep or equivalent machine.

After washing the cells in PBS ressuspend in the Lysis buffer and transfer to a new tube with the glass beads. Break up cells with Fast Prep, 8 x 60sec at 6m/sec with 5min on ice between the runs; spin down 1min full speed 4°C after FastPrep round 4 and 6. Then proceed with your protocol of choice.

I hope that was useful. Cheers.

Total (genomic + plasmid) DNA isolation from H. polymorpha; Mini-prep

Published in:   Sherman (1986); Methods in yeast genetics.

Miller, S.A., Dykes, D.D. and Polesky, H.F. A simple salting out procedure for extracting DNA from human nucleated cells. NAR 16 (3), 1215.

Goal:              Isolation of approximately 50 mg total (genomic + plasmid) H. polymorpha DNA for Southern blot analysis or plasmid rescue in E.coli.

Materials:       1. 

                     2. 

Solutions:       1.  YPD; 1% Yeast extract, 1% Bacto-peptone, 1% Glucose

2.   T100E50 (pH 8), 100 mM Tris-HCl, 50 mM EDTA, pH 8

3.   b-mercaptoethanol (supplied as 14.3 M)

4.   C100E10, 100 mM Na-Citrate buffer (pH 5.8), 10 mM EDTA

5.   10% SDS

6.   6 M NaCl (saturated)

7.   T10E1 (pH 8); 10 mM Tris-HCl, 1 mM EDTA, pH 8

8.   pretreated RNase 20 mg/ml (1000x) b)

Method:          1.  Grow 2.5 ml H. polymorpha cells overnight to saturation in YPD.

2.   Pellet cells; 2 min. at 10,000 rpm in eppendorf centrifuge

3.   Resuspend cells in  500 ml T100E50 (pH 8) + 5ml b-mercaptoethanol

4.   Incubate 15 min. at 37oC with shaking

5.   Pellet cells; 2 min. at 10,000 rpm in eppendorf centrifuge

6.   Resuspend (Wash) cells in 1 ml C100E10 (pH 5.8)

7.   Pellet cells; 2 min. at 10,000 rpm in eppendorf centrifuge

8.   Resuspend cells in 300 ml C100E10 (pH 5.8) with 1 mg Zymolyase

9.   Incubate 60 min at 37 oC

10. Add 15 ml 10% SDS, mix by inversion and incubate 10 min at 65 oC

11. aAdd 100 ml 6 M NaCl, mix by inversion and incubate 5 min on ice

12. aMix again and centrifuge 3 min at 14,000 rpm in eppendorf centrifuge

13. aTransfer supernatant containing the DNA to a clean 1.5 ml epp. tube

14. aAdd 300 ml isopropanol and gently mix by inversion

15. aCentrifuge 3 min at 14,000 rpm in eppendorf centrifuge

16. aCarefully decant the supernatant

17. Wash the pellet with 750 ml 70% ethanol

18. Centrifuge 3 min at 14,000 rpm in eppendorf centrifuge

19. Completely remove supernatant & air-dry the pellet (brief speed-vac spin is also OK)

20. Dissolve the DNA in 50 ml T10E1 (pH 8) with Rnaseb). (1 h at 50 oC or overnight at 4 oC)

21. Use 5-10 ml for restriction and Southern blot analysis

     Use 1-2 ml for E.coli transformation

Hi, There are different procedures for Candidal DNA extraction depends on the quality of the DNA and their intended use and type of sample. Rapid boiling and freezing will be enough to get the crud DNA from pure Candida culture but with poor quality and it is not beneficial in some uses. there are different kits and cheap manual procedures to extract DNA from fungi, choosing any of them depends on the experience and Lab. capabilities and available facilities ……

the following links will be helpful:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1248488/ source

https://www.diva-portal.org/smash/get/diva2:131034/FULLTEXT01.pdf

http://www.protocol-online.org/biology-forums-2/extraction.html

Thanks Mr Arjen for the detail protocol