RonDavis:YeastTransformation (Uli): Difference between revisions
(Yeast transformation using Lithium Acetate) |
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1 inoculate an overnight culture (5 ml) and grow to saturation | 1. inoculate an overnight culture (5 ml) and grow to saturation<br style="clear:both;"/> | ||
2 inoculate 1 ml of the saturated culture into 10 ml of fresh YPD and grow for 2 to 6 hours | 2. inoculate 1 ml of the saturated culture into 10 ml of fresh YPD and grow for 2 to 6 hours<br style="clear:both;"/> | ||
3 transfer 1 ml into a 1.5 ml Eppendorf tube (this is enough for 4 transformations) | 3. transfer 1 ml into a 1.5 ml Eppendorf tube (this is enough for 4 transformations)<br style="clear:both;"/> | ||
4 spin for 2 min at 8000 rpm | 4. spin for 2 min at 8000 rpm<br style="clear:both;"/> | ||
5 wash with 1 ml of 100 mM LiAc | 5. wash with 1 ml of 100 mM LiAc<br style="clear:both;"/> | ||
6 spin for 2 min at 8000 rpm | 6. spin for 2 min at 8000 rpm<br style="clear:both;"/> | ||
7 wash with 1 ml of 100 mM LiAc | 7. wash with 1 ml of 100 mM LiAc<br style="clear:both;"/> | ||
8 resuspend in | 8. resuspend in 80 μL Salmon Sperm DNA (boil for 10 min and then incubate on ice prior to use)<br style="clear:both;"/> | ||
9 aliquot 20 | 9. aliquot 20 μL of cell suspension into four 1.5 ml Eppendorf tube<br style="clear:both;"/> | ||
10 add DNA to be transformed (PCR product, plasmid, or vector + insert) | 10. add DNA to be transformed (PCR product, plasmid, or vector + insert)<br style="clear:both;"/> | ||
11 incubate for 30 min at | 11. incubate for 30 min at 30°C<br style="clear:both;"/> | ||
12 resuspend the pellet in 270 | 12. resuspend the pellet in 270 μL PEG (48%)<br style="clear:both;"/> | ||
13 add 30 | 13. add 30 μL of 1 M LiAc<br style="clear:both;"/> | ||
14 incubate for 30 min at | 14. incubate for 30 min at 30°C degrees C<br style="clear:both;"/> | ||
15 heat-shock for 30 - 60 min in water bath at | 15. heat-shock for 30 - 60 min in water bath at 42°C degrees C<br style="clear:both;"/> | ||
16 spin for 30 sec at 14000 rpm | 16. spin for 30 sec at 14000 rpm<br style="clear:both;"/> | ||
17 remove supernatant | 17. remove supernatant<br style="clear:both;"/> | ||
18a if selecting for a dominant drug-marker(such as KanMX) resuspend the pellet in 5 ml of YPD and incubate overnight at room temperature | 18a. if selecting for a dominant drug-marker(such as KanMX) resuspend the pellet in 5 ml of YPD and incubate overnight at room temperature<br style="clear:both;"/> | ||
18b if selecting for an auxotrophic marker (such as URA3, LEU2, HIS3) no recovery in YPD is needed | 18b. if selecting for an auxotrophic marker (such as URA3, LEU2, HIS3) no recovery in YPD is needed<br style="clear:both;"/> | ||
19 plate onto selective media and incubate at | 19. plate onto selective media and incubate at 30°C<br style="clear:both;"/> | ||
20 colonies should appear after ~ 2 days | 20. colonies should appear after ~ 2 days<br style="clear:both;"/> | ||
Latest revision as of 17:02, 7 October 2013
1. inoculate an overnight culture (5 ml) and grow to saturation
2. inoculate 1 ml of the saturated culture into 10 ml of fresh YPD and grow for 2 to 6 hours
3. transfer 1 ml into a 1.5 ml Eppendorf tube (this is enough for 4 transformations)
4. spin for 2 min at 8000 rpm
5. wash with 1 ml of 100 mM LiAc
6. spin for 2 min at 8000 rpm
7. wash with 1 ml of 100 mM LiAc
8. resuspend in 80 μL Salmon Sperm DNA (boil for 10 min and then incubate on ice prior to use)
9. aliquot 20 μL of cell suspension into four 1.5 ml Eppendorf tube
10. add DNA to be transformed (PCR product, plasmid, or vector + insert)
11. incubate for 30 min at 30°C
12. resuspend the pellet in 270 μL PEG (48%)
13. add 30 μL of 1 M LiAc
14. incubate for 30 min at 30°C degrees C
15. heat-shock for 30 - 60 min in water bath at 42°C degrees C
16. spin for 30 sec at 14000 rpm
17. remove supernatant
18a. if selecting for a dominant drug-marker(such as KanMX) resuspend the pellet in 5 ml of YPD and incubate overnight at room temperature
18b. if selecting for an auxotrophic marker (such as URA3, LEU2, HIS3) no recovery in YPD is needed
19. plate onto selective media and incubate at 30°C
20. colonies should appear after ~ 2 days
