Miniprep

1. Resuspend pelleted bacterial cells in 250 µl Buffer PI and transfer to a microcentrifuge tube.
2. Add 250 µl Buffer P2 and gently invert tube 4-6 times to mix
3. Add 350 µl Buffer N3 and invert immediately 4-6 times
4. Centrifuge for 10 min at 13,000 rpm
5. Add supernatant to QIAprep spin column
6. Centrifuge for 30-60s - Discard flow through
7. Wash QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60s - Discard flow through
8. Wash column by adding 0.75 ml Buffer PE and centrifuginh for 30-60s
9. Discard flow through and centrifuge for an additional 1 min to remove residual wash buffer
10. Place column in clean 1.5 ml microcentrifuge tube. To elute DNA add 50 µl water or buffer EB, let stand for 1 min and centrifuge for 1 min.

PCR Purification

1. 5 volumes of Buffer PB (with ethanol) to 1 volume PCR sample
2. Apply sample to column and spin for 1 min
3. Discard flow through
4. To wash add 0.75 ml of Buffer PE to column and centrifuge for 1 min
5. Discard flow through and place column back in the same tube
6. Cetrifuge for an addtitional minute (Dry Spin)
7. Place column in clean 1.5 ml tube
8. [Elute DNA] Add 30µl water to column, let stand for 1 min and centrifuge for 1 min

Gel Extraction

QlAquick Gel Extraction Kit

1. Cut Gel
2. Weigh it in a colorless tube
3. Add 3 volumes Buffer Q G to 1 volime Gel (100mg ~ 100µl)
4. Incubate @ 50° C for 10 min or until completely dissolved (vortex every 2-3 min to help dissolve)
5. Add 1 gel volume isopropanal to the sample and mix
6. Place a QlAquick soin column in a provided 2ml collection tube
7. Place sample in column & spin for 1 min --> discard flow through
8. To wash add 0.75 ml Buffer PE to column & centrifue for 1 min, then dry spin
9. Place column in 1.5 ml tube
10. Add 35µl H2O & centrifuge for 1 min.

E. Coli Transformation (Digestion/Ligation)

 
- 10 µl ligation
- 50 µl competent cells
--> 30m on ice
--> 45s heatshock 42° C
--> 2m on ice
- 250 µl of SOC media
--> 1h shake 37° C
                    

Yeast Transformation

• PEG = viscous, pipette slow
• boil ssDNA aliquots

Previous Day : Grow yeast strain to be transformed in 5-10 mL YPD overnight at 30° C

1. Set up digest to linearize DNA
2. Dilute O/N culture ~ 1:20 in YPD grow 2-4 hours at 30° C
3. Prepare ssDNA - boil for 10m cool on ice for at least 10m (10 µl of 10 mg/mL stock per transformation)
4. Harvest cells in centrifuge - 3000 rpm, 2-5 min
5. Wash with 1 ml 0.1 M LiOAin TE
6. Pellet cells - 3000 rpm , 2-5 min
7. Resuspend pellet in 100 µl 0.1 M LiOAc in TE per 2.5 ml culture, split into 100 µl per epindorph tube for each transformation
8. to 100 µl cells add 100 µg ssDNA (10 µl of 10 mg/mL sock), 1-5 µl target DNA
9. Add (in order): 480 µl 50% PEG 3350, 60 µl 10X TE, 60 µl 1 M LiOAc (for final 40% PEG, 1X TE, 0.1 M LiOAc) Optional: Add 75 µl DMSO definitely did
10. Vortex
11. Incubate 42° C for 30m & begin drying plates
12. Pellet (6000 rpm - 2m), discard supernatant (remove PEG completely by pipetting), resuspend in 500 µl YPD (or selective media)
13. Pellet, discard supernatant, resuspend in risidual ~ 50 µl YPD
14. Plate on selective media
15. Incubate 1-3 days

Colony PCR for screening E. Coli

Pick single colonies ( 5 or so from each plate ) mix in 25 µl H2O in a tube. Use 5 µl in PCR reaction

Cycle (Varies):

95° C | 5m

   30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever

load 5 µl onto gel

for all positive bands - take the rest of the bands and inoculate them into an overnight LB (+antibiotic) for miniprep

Colony PCR for Yeast & Patching

1. Number colonies
2. Patch on plate
3. Mix in 10 µl NaOH
4. Boil for 20m
5. PCR

Cycle (varies):

95° C | 5m
30x:
        95° C | 45s
        55° C | 30s
        72° C | 1m per kb

72° C | 10m 
 4° C | Forever
                    

Frozen Glycerol Stocks Yeast

1. Grow Overnight in YPD (2-5 mL) then dilute 1:20 in YPD, grow to OD 0.4-0.5
2. Add 350 µl cells to 350 µl sterile 60% glycerol in cryovial, vortex to mix, snap freeze in liquid nitrogen and store at -80° C (but actually we just stuck it in the freezer because nobody will let us use liquid nitrogen).