Team:Goettingen/protocol Plasmid Tran

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         <br /><br />
         <br /><br />
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             <p>Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a 1.5 mL tube. Gently add ~100 μl of competent cells. <br />
+
             <p>
-
             Incubate for 30 min on ice.<br />
+
            - Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a 1.5 mL tube. Gently add ~100 μl of competent cells. <br />
-
             Heat shock for 2 min @ 42°C. Put back on ice.<br />
+
             - Incubate for 30 min on ice.<br />
-
             Add 900 μl of LB to tubes. Incubate @ 37°C for 30 min.<br />
+
             - Heat shock for 2 min @ 42°C. Put back on ice.<br />
-
             Plate different concentrations (e.g. 50, 100, rest μL) of the cells on LB + antibiotics plates. Incubate them @ 37°C overnight.<br /><br />
+
             - Add 900 μl of LB to tubes. Incubate @ 37°C for 30 min.<br />
 +
             - Plate different concentrations (e.g. 50, 100, rest μL) of the cells on LB + antibiotics plates. Incubate them @ 37°C overnight.<br /><br />
             </p>
             </p>
             <h2 id="Y_tran">Yeast transformation</h2>
             <h2 id="Y_tran">Yeast transformation</h2>
-
             <p>Pick a colony into 5 ml YPAD medium (pH 5.8) and incubate overnight at 30°C with shaking. Until OD600 >1.5. <br />
+
             <p>
-
             Sub-culture 1 ml into 50 ml YPAD and grow at 30°C with shaking for 3.5-4h. Until OD600 0.6-1.2. <br />
+
            - Pick a colony into 5 ml YPAD medium (pH 5.8) and incubate overnight at 30°C with shaking. Until OD600 >1.5. <br />
-
             Collect cells by low speed centrifugation (4000 rpm, 5 min, room temperature) <br />
+
             - Sub-culture 1 ml into 50 ml YPAD and grow at 30°C with shaking for 3.5-4h. Until OD600 0.6-1.2. <br />
-
             Discard the medium and resuspend the cells by vortexing in 25 ml dH2O.<br />
+
             - Collect cells by low speed centrifugation (4000 rpm, 5 min, room temperature) <br />
-
             Respin cells (4000 rpm, 5 min, room temperature), discard supernatant and resuspend in 1 ml sterile dH2O.<br />
+
             - Discard the medium and resuspend the cells by vortexing in 25 ml dH2O.<br />
-
             Transfer to a 1.5 mL tube and respin at top speed for 10 sec to pellet cells. <br />
+
             - Respin cells (4000 rpm, 5 min, room temperature), discard supernatant and resuspend in 1 ml sterile dH2O.<br />
-
             Resuspend in 550 μl 100 mM LiAc pH7.5 and transfer 50 μl aliquots to 11 sterile microcentrifuge tubes. <br />
+
             - Transfer to a 1.5 mL tube and respin at top speed for 10 sec to pellet cells. <br />
-
             Centrifuge for 10 s at top speed to pellet cells, and remove the supernatant. <br />
+
             - Resuspend in 550 μl 100 mM LiAc pH7.5 and transfer 50 μl aliquots to 11 sterile microcentrifuge tubes. <br />
-
             To each tube add, in order: 240 μl 50% PEG 4000, 36 μl 1 M LiAc, 10 μl single-stranded DNA  (Salmon sperm, Invitrogen). <br />
+
             - Centrifuge for 10 s at top speed to pellet cells, and remove the supernatant. <br />
-
             0.5-1 μl plasmid DNA (250-500 ng of each plasmid) <br />
+
             - To each tube add, in order: 240 μl 50% PEG 4000, 36 μl 1 M LiAc, 10 μl single-stranded DNA  (Salmon sperm, Invitrogen). <br />
-
             Resuspend and mix thoroughly by pipetting or vigorous vortexing. Incubate at 42°C for 25 - 45 min. <br />
+
             - 0.5-1 μl plasmid DNA (250-500 ng of each plasmid) <br />
-
             Centrifuge cells at low speed (4000 rpm, 10 sec), remove medium and resuspend cell pellet in 1 ml YPAD for 1 h.<br />
+
             - Resuspend and mix thoroughly by pipetting or vigorous vortexing. Incubate at 42°C for 25 - 45 min. <br />
-
             Centrifuge cells at low speed (4000 rpm, 1 min) remove medium and resuspend cell pellet in 200 μl sterile dH2O. <br />
+
             - Centrifuge cells at low speed (4000 rpm, 10 sec), remove medium and resuspend cell pellet in 1 ml YPAD for 1 h.<br />
-
             Spread aliquots onto SC dropout medium (spread gently with spreading bar or using sterile glass beads). Allow to air-dry and incubate at 30°C for 2-3 days for colonies to grow.<br /><br />
+
             - Centrifuge cells at low speed (4000 rpm, 1 min) remove medium and resuspend cell pellet in 200 μl sterile dH2O. <br />
 +
             - Spread aliquots onto SC dropout medium (spread gently with spreading bar or using sterile glass beads). Allow to air-dry and incubate at 30°C for 2-3 days for colonies to grow.<br /><br />
             </p>
             </p>
         </div>
         </div>
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Revision as of 18:06, 15 September 2014

E.coli transformation



- Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a 1.5 mL tube. Gently add ~100 μl of competent cells.
- Incubate for 30 min on ice.
- Heat shock for 2 min @ 42°C. Put back on ice.
- Add 900 μl of LB to tubes. Incubate @ 37°C for 30 min.
- Plate different concentrations (e.g. 50, 100, rest μL) of the cells on LB + antibiotics plates. Incubate them @ 37°C overnight.

Yeast transformation

- Pick a colony into 5 ml YPAD medium (pH 5.8) and incubate overnight at 30°C with shaking. Until OD600 >1.5.
- Sub-culture 1 ml into 50 ml YPAD and grow at 30°C with shaking for 3.5-4h. Until OD600 0.6-1.2.
- Collect cells by low speed centrifugation (4000 rpm, 5 min, room temperature)
- Discard the medium and resuspend the cells by vortexing in 25 ml dH2O.
- Respin cells (4000 rpm, 5 min, room temperature), discard supernatant and resuspend in 1 ml sterile dH2O.
- Transfer to a 1.5 mL tube and respin at top speed for 10 sec to pellet cells.
- Resuspend in 550 μl 100 mM LiAc pH7.5 and transfer 50 μl aliquots to 11 sterile microcentrifuge tubes.
- Centrifuge for 10 s at top speed to pellet cells, and remove the supernatant.
- To each tube add, in order: 240 μl 50% PEG 4000, 36 μl 1 M LiAc, 10 μl single-stranded DNA (Salmon sperm, Invitrogen).
- 0.5-1 μl plasmid DNA (250-500 ng of each plasmid)
- Resuspend and mix thoroughly by pipetting or vigorous vortexing. Incubate at 42°C for 25 - 45 min.
- Centrifuge cells at low speed (4000 rpm, 10 sec), remove medium and resuspend cell pellet in 1 ml YPAD for 1 h.
- Centrifuge cells at low speed (4000 rpm, 1 min) remove medium and resuspend cell pellet in 200 μl sterile dH2O.
- Spread aliquots onto SC dropout medium (spread gently with spreading bar or using sterile glass beads). Allow to air-dry and incubate at 30°C for 2-3 days for colonies to grow.

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