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Yeast Genomic DNA preparation


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4 replies to this topic

#1 NickYang

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Posted 10 December 2010 - 01:10 PM

Hey guys,

I was doing a genomic dna prep and I accidentally added TE (step7) too early before the lysis step (step4-6).
Would my prep still work?

heres my protocol:
1. Grow 5ml cells o/n.
2. Centrifuge cells
3. Add o.5ml H2O and transfer cells to a screw-cap tube.
4. Add 0.2ml Genomic DNA buffer (2% Trition X-100, 1% SDS, 100mM NaCl, 10mM Tris-Cl (pH 8), 1mM Na2 EDTA)
5. Add 0.2ml PCA (phenol:chloroform:isoamyl alcohol (25:24:1))
6. Vortex for 4min.
7. Add 0.2ml TE
8. Centrifuge.
9. Tansfer the top, clear layer to a clean tube.
10. Add 100ml 100% isopropanol.
11. Leave @RT for 1hr.
12. Centrifuge
13. Add 0.4ml TE to resuspend.
14. Add 20ul 10M LiCl
15. Add 1ml 100% isopropanol
16. Leave @RT for 1hr.
17. Centrifuge.
18. Resuspend with 0.1ml TE

Thanks

#2 perneseblue

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Posted 10 December 2010 - 09:57 PM

should be okay. Is the volume still 0.5ml? Are you at step 3 and added 0.2ml TE by mistake? If so, just spin down the cells, remove the TE and resuspend cells in ).5ml water (step 3). TE is just 10mM Tris-HCl and 1mM EDTA
May your PCR products be long, your protocols short and your boss on holiday

#3 NickYang

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Posted 12 December 2010 - 02:11 AM

I was at step 6. but i added 0.2ml TE, which was step 7, before step 6.
So I had a mixture of 0.2ml TE, 0.2 Genomic DNA buffer, 0.2ml PCA, 0.3g glass beads for vortex lysis.

I am afraid that the lysis would be less efficient making the yield lower than expected.

#4 phage434

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Posted 12 December 2010 - 08:08 AM

You could just add a bit more phenol/chloroform in step 5 and continue. I was concerned that I didn't think things would lyse, but I see now that you are doing a glass bead purification. Don't forget the beads. Also, I assume you mean 100 ul not 100 ml of isopropanol. Just checking.

#5 NickYang

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Posted 13 December 2010 - 10:15 AM

You could just add a bit more phenol/chloroform in step 5 and continue. I was concerned that I didn't think things would lyse, but I see now that you are doing a glass bead purification. Don't forget the beads. Also, I assume you mean 100 ul not 100 ml of isopropanol. Just checking.


oops, 1ml isopropanol actually.

The result showed up with lower yield but looked fine.

Thx Perny and Phage.




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