Line 28: | Line 28: | ||
<li><b>Colony-PCR using Alkaline PEG</b> | <li><b>Colony-PCR using Alkaline PEG</b> | ||
<li style=" padding-left: 0.5cm; color:Black;"><b>Preparation of alkaline PEG reagent:</b></li> | <li style=" padding-left: 0.5cm; color:Black;"><b>Preparation of alkaline PEG reagent:</b></li> | ||
− | <ol type="1 | + | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > |
<li style=" padding-right: 0.3cm;"><span>Combine <b>60 g PEG 200</b> (Sigma-Aldrich or equivalent) with <b>0.93 mL 2 M KOH </b>and <b>39 mL water</b>. If desired, KOH can be substituted by NaOH in the reagent. | <li style=" padding-right: 0.3cm;"><span>Combine <b>60 g PEG 200</b> (Sigma-Aldrich or equivalent) with <b>0.93 mL 2 M KOH </b>and <b>39 mL water</b>. If desired, KOH can be substituted by NaOH in the reagent. | ||
<br/><span style="color: #7fb983;">Note:</span> PEG 200 is measured by mass rather than volume because of the viscosity of the liquid.</span></li> | <br/><span style="color: #7fb983;">Note:</span> PEG 200 is measured by mass rather than volume because of the viscosity of the liquid.</span></li> | ||
Line 35: | Line 35: | ||
</ol> | </ol> | ||
<li style=" padding-left: 0.5cm; color:Black;"><b>Execution of the Colony PCR:</b></li> | <li style=" padding-left: 0.5cm; color:Black;"><b>Execution of the Colony PCR:</b></li> | ||
− | <ol type="1 | + | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > |
<li style=" padding-right: 0.3cm;"><span>Pick a bit of cell material of colony with a pipette tip and put it in a PCR tube with <b>50 µL</b> of the <b>reagent</b>. Keep the pipette tip there during incubation time.</span></li> | <li style=" padding-right: 0.3cm;"><span>Pick a bit of cell material of colony with a pipette tip and put it in a PCR tube with <b>50 µL</b> of the <b>reagent</b>. Keep the pipette tip there during incubation time.</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>Lyse the samples by incubation them in the prepared PEG reagent for <b>3-15 min</b> at room temperature.</span></li> | <li style=" padding-right: 0.3cm;"><span>Lyse the samples by incubation them in the prepared PEG reagent for <b>3-15 min</b> at room temperature.</span></li> | ||
Line 44: | Line 44: | ||
<li><b>Colony-PCR with Heatshock</b> | <li><b>Colony-PCR with Heatshock</b> | ||
− | <ol type="1 | + | <ol type="1"style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > |
<li style=" padding-right: 0.3cm;"><span>Pick colonies from the transformed plates.</span></li> | <li style=" padding-right: 0.3cm;"><span>Pick colonies from the transformed plates.</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>Streak one part on a masterplate.</span></li> | <li style=" padding-right: 0.3cm;"><span>Streak one part on a masterplate.</span></li> | ||
Line 57: | Line 57: | ||
<span style="color: #7fb983;">Aim:</span>find a colony which carries the correct insert in the vector. | <span style="color: #7fb983;">Aim:</span>find a colony which carries the correct insert in the vector. | ||
</span></li> | </span></li> | ||
− | <ol type="1 | + | <ol type="1"style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > |
<li style=" padding-right: 0.3cm;"><span>Pick colonies of your plates. | <li style=" padding-right: 0.3cm;"><span>Pick colonies of your plates. | ||
<br/> | <br/> | ||
Line 165: | Line 165: | ||
<ol class="protocolhide" style=" padding-left:0.1cm; padding-right:0.1cm;list-style-type: none;"> | <ol class="protocolhide" style=" padding-left:0.1cm; padding-right:0.1cm;list-style-type: none;"> | ||
<li ><b>E. coli</b> | <li ><b>E. coli</b> | ||
− | <ol type="1 | + | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm;align-content: center;"> |
<li style=" padding-right: 0.3cm;"><span>Thaw 100 µL <b>E. coli DH5α</b> or <b>E. coli BL21(DE3)</b> Gold on ice</span></li> | <li style=" padding-right: 0.3cm;"><span>Thaw 100 µL <b>E. coli DH5α</b> or <b>E. coli BL21(DE3)</b> Gold on ice</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>Add <b>DNA</b> (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; | <li style=" padding-right: 0.3cm;"><span>Add <b>DNA</b> (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; | ||
Line 175: | Line 175: | ||
incubate at <b>37°C at 250rpm for 45 min </b>(45-60 min) for recovery of the cells.</span></li> | incubate at <b>37°C at 250rpm for 45 min </b>(45-60 min) for recovery of the cells.</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>Plate cells on <b>LB agar plate</b> with antibiotic respectively and dry them under the clean bench. | <li style=" padding-right: 0.3cm;"><span>Plate cells on <b>LB agar plate</b> with antibiotic respectively and dry them under the clean bench. | ||
− | <ol style="list-style-type:disc; padding- | + | <ol style="list-style-type:disc; padding-right: 0.1cm"> |
− | <li><span>200 µL</span></li> | + | <li style=" padding-right: 0.3cm;"><span>200 µL</span></li> |
− | <li><span>resuspended pellet (from centrifugation of the leftover)</span></li> | + | <li style=" padding-right: 0.3cm;"><span>resuspended pellet (from centrifugation of the leftover)</span></li> |
</ol> | </ol> | ||
</span></li> | </span></li> | ||
Line 184: | Line 184: | ||
</ol></li> | </ol></li> | ||
− | <li><b> | + | <li><b>in Saccharomyces cerevisiae</b> |
− | <ol type="1 | + | <ol type="1" style=" padding-left:0.1cm; padding-right:0.1cm; align-content: center" > |
<li style=" padding-right: 0.3cm;"><span>Combine 0.5 - 1 µg each of desired fragments, bring to a total volume of 34uL with <b>ddH2O</b> (for plasmids, use 100 - 300ng of plasmid DNA)</span></li> | <li style=" padding-right: 0.3cm;"><span>Combine 0.5 - 1 µg each of desired fragments, bring to a total volume of 34uL with <b>ddH2O</b> (for plasmids, use 100 - 300ng of plasmid DNA)</span></li> | ||
<li style=" padding-right: 0.3cm;"><span>2. Prepare <b>transformation mixture</b> (Can be scaled up and prepared as master mix):<br/>240 ul 50% PEG 3350<br/> | <li style=" padding-right: 0.3cm;"><span>2. Prepare <b>transformation mixture</b> (Can be scaled up and prepared as master mix):<br/>240 ul 50% PEG 3350<br/> | ||
Line 192: | Line 192: | ||
50ul single stranged carrier DNA | 50ul single stranged carrier DNA | ||
</span></li> | </span></li> | ||
− | <li style=" padding-right: 0.3cm;"><span> | + | <li style=" padding-right: 0.3cm;"><span>Centrifuge aliquot of competent cells at 16000xG for 15s.</b></span></li> |
− | + | <li style=" padding-right: 0.3cm;"><span>Resuspend cells in 34ul (Plasmid) <b>DNA solution.</b></span></li> | |
− | <li style=" padding-right: 0.3cm;"><span> | + | <li style=" padding-right: 0.3cm;"><span>Add transformation mixture and vortex gently.</span></li> |
− | <li style=" padding-right: 0.3cm;"><span> | + | <li style=" padding-right: 0.3cm;"><span>Incubate tubes at 30°C for 30 minutes.</span></li> |
− | <li style=" padding-right: 0.3cm;"><span> | + | <li style=" padding-right: 0.3cm;"><span>Invert tubes, then incubate for precisely 30 min at 42°C.</span></li> |
− | <ol type=" | + | <li style=" padding-right: 0.3cm;"><span>Centrifuge cells again at 16000xg for 15s.</span></li> |
− | <li><span> | + | <li style=" padding-right: 0.3cm;"><span>Resuspend pellet in 100ul dd H2O, spread on <b>selection medium</b> |
− | <li><span> | + | <br/><span style="color: #7fb983;">Note:</span>If <b>antibiotic marker</b> is used, then outgrowth is needed: |
+ | <ol type="a" style="padding-right: 0.1cm"> | ||
+ | <li style=" padding-right: 0.3cm;"><span>Resuspend cells in 500ul YPD medium and incubate for precisely 30 min at 42°C.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>centrifuge cells at 16000 x G, 15s.</span></li> | ||
+ | <li style=" padding-right: 0.3cm;"><span>resuspend cells on 100ul ddH2O, then spread on medium</span></li> | ||
+ | </ol></span></li> | ||
</ol> | </ol> | ||
− | + | </li> | |
− | + | ||
− | + | ||
− | + | ||
<li>Saccharomyces </li> | <li>Saccharomyces </li> |
Revision as of 17:57, 7 October 2016
Protocols
General
-
Polymerase Chain Reaction (PCR)
- Colony-PCR using Alkaline PEG
- Preparation of alkaline PEG reagent:
- Combine 60 g PEG 200 (Sigma-Aldrich or equivalent) with 0.93 mL 2 M KOH and 39 mL water. If desired, KOH can be substituted by NaOH in the reagent.
Note: PEG 200 is measured by mass rather than volume because of the viscosity of the liquid. - Adjust the pH to 13.3–13.5..
Note: Due to storage, some batches of PEG 200 have an acidic, rather than neutral pH. In this case, add an additional amount of alkali to reach the target pH range. - Execution of the Colony PCR:
- Pick a bit of cell material of colony with a pipette tip and put it in a PCR tube with 50 µL of the reagent. Keep the pipette tip there during incubation time.
- Lyse the samples by incubation them in the prepared PEG reagent for 3-15 min at room temperature.
- Use 1-5 µL of the lysates as template in a 20-50 µL PCR reaction.
Note: The aliquot of the sample lysate should not exceed 10% of the final volume of a PCR mixture.
- Colony-PCR with Heatshock
- Pick colonies from the transformed plates.
- Streak one part on a masterplate.
- Suspend the other part in 10 µL water.
- Incubate the suspended sample at 90°C for 5-6 minutes.
- Perform a PCR with primers that bind to the insert and with 1-5 µL of the lysate as template in a 20-50 µL PCR reaction.
- Extended Colony-PCR
- Aim:find a colony which carries the correct insert in the vector.
- Pick colonies of your plates.
- Streak a part of the colony on a master plate.
- Suspend the other part in master sample. (20 µL of ddH2O)
- Repeat step 1 eight times → eight colonies on each master plate and in each master sample.
- Repeat step 2 four times (or more) → four (or more) master plates/master samples.
- incubate the master plates at 37°C.
- Heat the master samples at 90°C for 6 minutes.
- Perform a Colony PCR with the master samples and with primers that anneal to the correct insert.
- Perform an agarose-gelelectrophoresis with the results from the colony PCR.
- If any sample shows a band with the correct size, then use this sample for the next step.
- Perform a Colony PCR with all eight colonies (taken from the master plate) that were part of the positive tested master sample.
- Perform another agarose-gelelectrophoresis and analysis with these samples.
- If any sample shows a band with the correct size, then use this sample for further experiments.
Note:This experiment allows you to test a huge amount of colonies at the same time, and then to identify the exact colony which carries the correct insert.
- TCA precipitation with Yeast cells
- Solutions
- SDS sample buffer:
2% (w/v) 2-Mercaptoethanol or 3.1% (w/v) DTT or 125 mM Tris
20% (v/v) Glycerol
0.001% (w/v) Bromophenol Blue
4% (w/v) SDS - 50% TCA: 50% (w/v) Trichloroacetic Acid
- NaOH/Mercaptoethanol Solution:
1% (v/v) 2-Mercaptoethanol
0.25 M NaOH
- SDS sample buffer:
- Grow yeast cells to a cell density of approximately an Optical Density at 600 nm (OD600) of 1 (1 X 107 cells/ml).
- Centrifuge the 1.5 ml of cells at 5,000 rpm in a microcentrifuge for 5 min.
- Remove the supernatant and resuspend the cells in 0.25 ml of NaOH/Mercaptoethanol Solution.
- Incubate the cells for 10 min on ice.
- Add 0.16 ml of 50% TCA.
- Incubate for 10 min on ice.
- Centrifuge at full speed in a microcentrifuge for 10 min.
- Remove the supernatant and resuspend the pellet in 1 ml of ice-cold Acetone.
- Centrifuge at full speed in a microcentrifuge for 10 min.
- Remove the supernatant and allow the pellet to air dry. Resuspend the pellet in 100-200 μl of SDS Sample Buffer.
- Proceed with SDS-PAGE and Western Blotting.
- Lysozym
- Centrifuge the samples for 4 min at 4°C and 14000 rpm.
- Freeze the "dried" pellet overnight.
- Add 150 µL buffer on the pellet, resuspend it (e.g. with LB-medium) and incubate for 5 min at room temperature.
- Add 150 µL lysozym solution (c = 5-8 mg/mL).
- Incubate the samples for 1 h at 37°C and 250 rpm.
- Centrifuge the samples for 4 min at 4°C and 14000 rpm.
- Decant the supernatant.
- Sonication
- conditions:
- time: 3 min
- pulse on: 30 s
- pulse off: 20 s
- amplitude: 50%
- Glass beads
- Put some small glass beads into your sample tubes.
- Vortex your samples (5-10 min at level 2-6), avoid foam on the top of your sample.
- Put the used beads into a waste bottle filling with 70% EtOH for.
- E. coli
- Thaw 100 µL E. coli DH5α or E. coli BL21(DE3) Gold on ice
- Add DNA (2-400 ng/µL: 50 ng if Plasmid DNA; 2 µL (~20 ng) ligation mixture; 1-4 µL PLICing-reaction) to the competent cells and swirl gently
- Incubate for 15-30 min on ice.
- Perform heatshock of the competent cells using a preheated water bath at <42°C for 45s (DH5α, BL21).
- Samples were immediately cooled down 5 min on ice.
- Fill up the transformation mix to 1 mL with SOC media and incubate at 37°C at 250rpm for 45 min (45-60 min) for recovery of the cells.
- Plate cells on LB agar plate with antibiotic respectively and dry them under the clean bench.
- 200 µL
- resuspended pellet (from centrifugation of the leftover)
- Incubate the agar plates at 37°C overnight(16-18 hours).
- Count single colonies of each agar plate on the next day.
- in Saccharomyces cerevisiae
- Combine 0.5 - 1 µg each of desired fragments, bring to a total volume of 34uL with ddH2O (for plasmids, use 100 - 300ng of plasmid DNA)
- 2. Prepare transformation mixture (Can be scaled up and prepared as master mix):
240 ul 50% PEG 3350
36ul 1M LiAc
50ul single stranged carrier DNA - Centrifuge aliquot of competent cells at 16000xG for 15s.
- Resuspend cells in 34ul (Plasmid) DNA solution.
- Add transformation mixture and vortex gently.
- Incubate tubes at 30°C for 30 minutes.
- Invert tubes, then incubate for precisely 30 min at 42°C.
- Centrifuge cells again at 16000xg for 15s.
- Resuspend pellet in 100ul dd H2O, spread on selection medium
Note:If antibiotic marker is used, then outgrowth is needed:- Resuspend cells in 500ul YPD medium and incubate for precisely 30 min at 42°C.
- centrifuge cells at 16000 x G, 15s.
- resuspend cells on 100ul ddH2O, then spread on medium
- Saccharomyces
Analytics
-
Gelelectrophoresis
-
- Carefully remove the comb of the prepared solid agarose gel.
- Put the gel into the gel-electrophoresis set up filled with buffer.
- Pipette 1 µL loading dye on a piece of parafilm.
- Add 5 µL of the sample to it and mix it by pipetting it up and down (avoid bubbles).
- Pipette the whole solution (6 µL), without air in the pipette tip, slowly into one well of the gel.
- Pipette 2.5 µL ladder into another well as a comparison (e.g. ladder 08: #SM0311).
- Let it run for 35 min: 300 mA, 90V, 50W.
- Take out the gel, analyze it and then discard the gel afterwards.
-
-
Analysis of Expression Level
- Preparation of pre-culture
- Fill 20 mL LB media into a (small) flask, add the corresponding antibiotics.
- Inoculate the pre-culture.
- Incubate your culture at 37°C, 250 rpm for 16-18 hours.
- Preparation of main culture
- Fill 200 mL LB media into a 1000 mL flask and add the corresponding antibiotics.
- Inoculate the main culture with 2-5 mL pre-culture, as to reach an OD600 of 0.1 in the main culture.
- Incubate your main culture at 37°C, 250 rpm till it reaches the OD600 of 1 (2-4 hours).
- Take a sample (500µL) out of your main culture and store it at -20°C.
- Induce the expression of your main culture by adding a final concentration of each 0.5 / 1 / 1.5 mM IPTG.
- Incubate at 37°C, 250 rpm for an hour.
- Take a sample hourly afterwards. (as described above)
- Analysis
- Perform a cell lysis with the samples which were taken and frozen at -20°C.
- Perform a Skim Milk Assay with every lysed sample.
- Perform a SDS-gel with all lysed samples.
- Preparation of pre-culture
-
SDS
-
Skim Milk Assay
-
AAPF (Alanin-Alanin-Prolin-Phenylalanin) Assay
Cloning
-
Enzymatic Digestion
-
Dephosphorylation
- Dephosphorylation after restriction
- Add 1 unit of rSAP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) after restriction (example: 1 µL for 2000 kb) and according buffer (volume according to concentration of buffer).
- Incubate at 37°C for 30–60 minutes.
- Stop reaction by heat-inactivation of rSAP at 65°C for 5 minutes.
- Store at -20°C.
- Dephosphorylation after restriction
-
Ligation
-
Site Directed Mutagenesis (SDM) and Site Saturation Mutagenesis (SSM)
Media
Devices
Kits