Allow the DNA replicates to grow massively confluent. The protocol says they should turn the media yellow every day for 4-5 days (feeding every day as usual). I have gotten sufficient DNA from colonies grown only two days, so there is clearly some leeway.
-Label all the dishes and lids to avoid confusion.
-Aspirate the medium and wash twice with 180 µL warm PBS.
-Apply 50 µL of freshly prepared lysis buffer to each well and cover the top with plastic-wrap. Put the lid back on and stack the dishes. Wrap the stack with plastic wrap and put into a closed, humid container, such as tupperware with wet paper towels. Put the humidified container at 50-60 degrees overnight.
RECIPE FOR LYSIS BUFFER:
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10 mM Tris-HCL, pH 7.5 |
0.1mL 1M Tris stock |
|
10 mM EDTA |
0.2 mL 0.5M EDTA stock |
|
10mM NaCl |
0.02mL 5M NaCl stock |
|
0.5% sarkosyl |
1.0 mL 5% stock of N-lauroylSarcosine sodium salt aka sarkosyl.(Sigma L-9150). Stock is prepared in sterile ddH2O with gentle mixing to dissolve–no other histrionics required. |
|
1mg/mL proteinase K |
0.5 mL 20mg/mL proteinase K stock. |
|
8.18 mL sterile ddH2O |
|
|
10 mL total (enough for 2 plates) |
-The next day gently remove the plastic wrap. Do not concern yourself with small amounts of condensation on the wrap.
-Apply 100 µL ethanol+ salt (10 mL of 95% ethanol+ 0.15 mL of 5M NaCl stock, put on ice) to each well. Put lid back on top. Do not mix vigorously–just squirt it on and go to the next row.
-Leave out on bench for 30-60 minutes, preferably on a dark piece of paper to visualize the DNA. Do not disturb the plates, knock them, or otherwise jar the solutions.
-DNA should be visible as a white filamentous network or a white sheet.
-Gently Invert the plates on a stack of paper towels.
-Wash three times with 150 µL 70% ETOH, inverting gently each time.
-After the last wash invert once more to remove trace ETOH (you can be a little rougher here–visualize the DNA as you go to see how you are doing).
-Tilt plates slightly and allow to air dry for 20 minutes. Do not over-dry the plates as this makes resuspension harder.
-Apply 20 µL of low EDTA TE to each well (10 mM Tris/0.1mM EDTA) and cover the plate with plastic wrap. Put the lid back on and stack your dishes, then wrap again for safe keeping. Store overnight in the fridge (good forever).
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