Transformation This protocol describes the Transformation of mutS cells.
It applies equally for DH5a cell transformation.

Transformation

1. Preheat waterbath to 42 C.
2. Add 5-10 uL of Plasmid DNA to a 1.5 mL microfuge tube that
contains 200 uL of competent BMH 71-18 mutS cells.
(Note: Do not add a volume of Plasmid DNA that exceeds
10% of the volume of the competent cells.) 3. Incubate on ice for 20 min.
4. Place tubes in the 42 C waterbath for 90 sec.
5. Add 1 mL LB (without antibiotic) to each tube.
6. Incubate at 37 C for 1 h on invertor.
7. Add 4 mL of LB that contains 50 ug of ampicillin
per mL of LB to 14 mL Falcon tubes.
8. Add the 1.2 mL recovery mixes from step 6 to
a tube that contains the LB with Ampicillin.
9. Incubate for 12 h at 37 C on a rotator/shaker at 220 rpm.

Once the 12 h incubation is completed, take these 5 mL
overnight cultures through the mini-prep protocol.

Fan's Revised Mini-Prep Protocol

1. Spin down overnight cultures in Sorval for 5 minutes @ 5,000 rpm.
2. Remove the supernatant via aspiration.
3. Add 0.5 mL solution I. Re-suspend the pellet and transfer the suspension
to clean 1.5 mL eppendorf microfuge tubes.
4. Centrfuge the tubes 15,000 rpm @ 4C, 2 min.
5. Remove the supernatant via aspiration.
6. Add 100 uL of solution I and re-suspend the pellet by pipetting.
7. Make solution II: 7mL H2O + 2mL (1M) NaOH + 1 mL (10%) SDS.
8. Add 200 uL of solution II. Use the pipettor to mix
unless the solution is clear.
9. Add 150 uL of solution III to each tube. Mix a few times
via inversion times. Leave at room temperature for 5-10 minutes.
10. Centrifuge @ 4C for 10-15 minutes.
11. Remove the supernatant to a clean microfuge tube.
12. Add 1 mL of ice-cold 100% EtOH.
13. Leave on ice 30 minutes.
14. Centrifuge @ 15,000 rpm @ 4C for 10-15 minutes.
15. Remove the supernatant via aspiration.
16. Add 200 uL of TE/RNAse. (each sample should have 10-20 ug of total RNAse;
This means add ~10 uL of (10 mg/mL stock) RNAse per mL of TE.
17. Incubate 1 h @ 37C.
18. Add 1 volume of Phenol and 1 volume of CHCl3/isoamyl alcohol
(12:1, v/v), and vortex tubes.
19. Centrifuge 5 minutes @ 15,000 rpm.
20. Remove supernatant to a clean microfuge tube.
21. Add 1 mL 100% EtOH and 60 uL Na Acetate (3M, pH 5.2).
22. Store overnight @ -20C or for 20-30 minutes on dry ice to precipitate the DNA.
23. Centrifuge 15,000 rpm @ 4C for 10-15 minutes to collect precipitate.
24. Remove supernatant via aspiration.
25. Dissolve pellet in 20-50 uL of TE.