Some one please help, I have amplified a 2.3 kb fragment (contains TAP and Kanmx cassete) pYM13 was used as template with overlapping primers. I followed this paper (https://www.ncbi.nlm.nih.gov/pubmed/15334558) for the experiment.
After transformation Can i do selection on G418 plates?
Do i need to transform in haploid cells or diploid cells? and Why?
yes, you can use G418 plate, but to be known that you may not get a clean plate with positive colonies, instead you will get plenty of tiny colonies, with some very big colonies standing out after 4-7 days post transformation. Try to confirm from the big colonies.
Better to use haploid, simple and more direct. Below is the protocol I used for many years. Work everytime.
High Efficiency Transformation Protocol
Day 1 (for 10 transformations)
Inoculate the yeast strain into 5 ml of liquid medium (2x YPAD or SC selection medium) and incubate overnight on a rotary shaker at 200 rpm and 30°C. Place a bottle of double strength YPAD broth (2x YPAD) and a 250 ml culture flask in the incubator as well.
Day 2
1. Determine the titer of the yeast culture by pipetting 10 ul of cells into 1.0 ml of water in a spectrophotometer cuvette and measuring the OD at 600 nm. For many yeast strains a suspension containing 1 x 106 cells/ml will give an OD600 of 0.1.
2. 1 ml to 100 ml inoculate with fresh YPD, grow at 30C for 4 hrs at 30C 200 RPM.
Transfer 50 ml of the pre-warmed 2x YPAD to the pre-warmed culture flask and add 2.5 x 108 cells to give 5 x 106 cells/ml.
3. Incubate the flask on a rotary or reciprocating shaker at 30°C and 200 rpm.
4. When the cell titer is at least 2 x 107 cells/ml, which should take about 4 hours, harvest the cells by centrifugation at 3000 g for 5 min, wash the cells in 25 ml of sterile water and resuspend in 1 ml of sterile water.
5. Boil a 1.0 ml sample of carrier DNA for 5 min and chill in an ice/water bath while harvesting the cells.
6. Transfer the cell suspension to a 1.5 ml microcentrifuge tube, centrifuge for 30 sec and discard the supernatant.
7. Add water to a final volume of 1.0 ml and vortex mix vigorously to resuspend the cells.
8. Pipette 100 µl samples (ca. 108 cells) into 1.5 ml microfuge tubes, one for each transformation, centrifuge at top speed for 30 sec and remove the supernatant.
9. Make up sufficient Transformation Mix for the planned number of transformations plus one extra. Keep the Transformation Mix in ice/water.
Number of Transformations
Reagents
1
5 (6X)
10 (11X)
PEG 3500 50% w/v
240 µl
1440 µl
2640 µl
LiAc 1.0 M
36 µl
216 µl
396 µl
Boiled SS-carrier DNA
50 µl
300 µl
550 µl
Plasmid DNA plus Water
34 µl
204 µl
374 µl
Total
360 µl
2160 µl
3960 µl
10. Add 360 µl of Transformation Mix to each transformation tube and resuspend the cells by vortex mixing vigorously.
11. Incubate the tubes in a 42°C water bath for 40 (varies as strains diff) min.
12. Microcentrifuge at top speed for 30 sec and remove the Transformation Mix with a micropipettor.
For Kan G418 selection, add broth medium incubate in 30C shaking for 2 hrs. then spin down.
13. Pipette 1.0 ml of sterile water into each tube; stir the pellet by with a micropipette tip gentlely.
14. Plate appropriate dilutions of the cell suspension onto YPD with 300 mg/ml G418 for KanR selection
15. Incubate the plates at 30°C for 3 to 4 days and count the number of transformants.
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