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From 2011.igem.org

Contents 1 Lab Protocols 1.1 Clonning procedures 1.2 Background 1.3 Preparetion 1.4 Protocol 1.5 Gel Photo 1.6 Checklist Procedure

Lab Protocols

Clonning procedures

1. Getting the DNA parts
   
   
1. For parts in kit plates

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1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick^TM-standard part that you want.
2. Pipette 10 uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully re-suspended.
3. <a href="http://partsregistry.org/Help:Transformation_Protocol" target="_blank">Transform</a> 1 or 2 uL of the re-suspended DNA into desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.
4. Pick a single colony and inoculate LB medium (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to <a href="http://openwetware.org/wiki/Miniprep/Kit-free_high-throughput_protocol" target="_blank">miniprep</a> the DNA and make your own glycerol stock.
2. For new synthesized DNA parts
1. The DNA parts are sent in nmol values in each. These data are determined for all of them individually
2. As a stock solution preparation, these DNA parts are pipetted with PCR water or TE buffer which is 100 times of DNA amount. The final solution is prepared as 100uM.
3. For each transformation procedures, the 10uM of stock DNA part in water is transformed. 1uL is taken from stock DNA solution and is added to 9uL PCR water.
   
   
2. Preparation of Competent Cells
   
   
   1. Inoculate a single colony of E. Coli cells into 50 ml LB medium. Grow overnight at 37 C with moderate shaking (250rpm)
   2. Inoculate 4 mL of the culture into 400 mL LB medium in a sterile 2-liter flask. Grow at 37 C shaking at 250 rpm to an OD590 of 0.375.

      This procedure requires that cells be growing rapidly (early- or mid-log phase). Accordingly, it is very important that the growing cells have sufficient air. Overgrowth of culture (beyond OD590 of 0.4) decreases the efficiency of transformation.

   3. Aliquot culture into eight 50 mL prechilled, sterile polypropylene tubes and leave the tubes on ice 5 to 10 min.

      Cells should be kept cold for all subsequent steps.
      Larger tubes or bottles can be used to centrifuge cells if volumes of all subsequent solutions are increased in direct proportion.

   4. Centrifuge cells 7 min at 1600 g (3000 rpm in JS -5.2),  4 C.

   Allow centrifuge to decelerate without breake.
   We have not attempted to determine whether deceleration without braking is critical to this procedure. However, we do not use the breake for this step or for the subsequent centrifugation steps.

   5. Pour off subernant and resuspend each pellet in 10 ml ice-cold CaCl2 solution.Re-suspension should be performed very gently and all cells kept on ice.
   6. Cetrifuge cells 5 min at 1100 g, 4 C. Discard supernatant and re-suspend each pellet in 10 mL ice-cold CaCl2 solution. Keep re-suspend cells on ice for 30 min.
   7. Centrifuge cells 5 min at 1100 g, 4 C. Discard supernatant and re-suspend each pellet in 2 mL ice-cold CaCl2 solution.

      It is important to re-suspend this final pellet well. The suspension may be left on ice for several days.

   8. Dispense cells into prechilled, sterile polypropylene tubes (250 ml aliquots are convenient). Freeze immediately at -70 C.
3. Transformation
   
   
1. Aliquot 10 ng of DNA in a volume 10 to 25  mL into a sterile 15 mL round bottom test tube and place on ice.

Plasmid DNA can be used directly from ligation reactions. When this is done , more DNA is usually used. However, if there is <1 mg of DNA in the ligation reaction, or if the ligation reaction is from low gelling/melting temperature agarose, it is wise to dilute the ligation mix.

2. Rapidly thaw competent cells by warming between hands and dispense 100 mL immediately into test tubes containing DNA. Gently swirl tubes to mix, then place on ice for 10 min.

Competent cells should be used immediately after thawing. Remaining cells should be discarded rather than refrozen

3. Heat shock cells by placing tubes into a 42 C water bath for 2 min

Alternatively incubate at 37 C for 5 min.

4. Add 1 ml LB medium to each tube. Place each tube on a roler drum at 250 rpm for 1 hr at 37 C.
5. Plate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.

It is convenient to plate several different dilutions of each transformation culture. The remainder of the mixture can be stored at 4 C for subsequent platings.

6. When plates are dry, incubate 12 to 16 hr at 37 C.
4. Plasmid isolation

This procedure is performed with Fermentas GeneJET™ Plasmid Miniprep Kit

1. Pick a single colony
2. Inoculate in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented with the appropriate selection antibiotic.
3. Incubate at 225 rpm not longer than 12-14 h at 37 C .
4. Centrifuge at 4000 rpm for 5 min at 4 C .
5. Discard the supernatant and keep pellet.
6. Resuspended  the pellet with 250 ul Resuspension solution. (Bacteria should be resuspended completely by vortexing until no cell clumps remain)
7. Transfer the cell suspension to eppendorf.
8. Add 250 uL Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes slightly clearPlate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.

Do not vortex!
Do not incubate for more than 5 min. (To avoid denaturation of supercoiled plasmid DNA!)

9. Add 350 uL Neutralization solutionand mix immediately and throughly by inverting the tube 4-6 times.

(The neutralized bacterial lysate is cloudy and viscous) ("throughly" to avoid localized precipitation of bacterial cell debris)

10. Centrifuge at 13000 rpm for 5 min to precipitate cell debris and chromosomal DNA.
11. Transfer the supernatant to spin column (about 600 uL).

(Avoid disturbing or transferring the white precipitate)

12. Centrifuge at 13000 rpm for 1 min.
13. Discard the flow-through.
14. Place the column back into same collection tube.
15. Add 500 uL Wash solution to spin column.
16. Centrifuge at 13000 for 1 min
17. Discard the flow-through.
18. Repeat this step with using 500 uL Wash solution.
19. Discard the flow-through
20. Centrifuge at 13000 rpm for an additional 1 min to remove residual Wash solution.
    (This step is essential to avoid residual ethanol in plasmid preps)
21. Transfer spin column into a fresh 1.5 mL epp.
22. Add 50 uL Elution buffer into center of the spin column membrane to elute the plasmid DNA

Do not contact the membrane with pipette tip!

23. Incubate for 2 min at 25 C. 

To increase yield incubation is done for 2 min at heat block at 42 C

24. Centrifuge at 13000 for 2 min.
25. Discard the column and store the purified plasmid DNA at -20 C.

OPTIONAL= additional elution step with elution buffer or water. This step increases the yield by 10-20 %.
NOTE:  For elution of plasmids ≥ 20 kb, prewarm Elution buffer to 70 C before applying.

5. Restriction digestion

This procedure is performed with NEB  BioBrick™ Assembly Kit

• Digest Upstream Part with EcoRI-HF and Spel
  500 ng Upstream Part Plasmid
  1 uL EcorI-HF
  1 uL Spe I
  5 uL 10*NE  Buffer 2
  0.5 uL 100* BSA
  To 50 uL add dH2O
  
  
• Digest downstream part will  Xbal and PstI
  500 ng Downstream Part Plasmid
  1 ul XbaI
  5 uL 10*NE Buffer 2
  0.5 uL 100* BSA
  To 50 uL add dH2O
  
  
• Digest the destination plasmid with EcorI-HF and Pst I:

The destination plasmid DNA should either be prepared with PCR or contain the toxic gene (e.g, ccdB, sacB) in the cloning site to avoid the need for gene purification.
The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.
500 ng Destination Plasmid DNA
1 uL EcorI- HF
1 uL PstI
15 uL 10* NE Buffer 2
0.5 uL 100*BSA
To 50 uL add dH2O
 
Incubate all 3 restriction digest reactions at 37 C for 10 min and then heat inactivate at 80 C for 20 min.

6. AGE (Confirmation)

Background

• EtBr stains only dsDNA. You cannot see ssDNA on gel
• Minimum amount of DNA load:

200 ng (Aysu)
• Each band of 1kb ladder is approx. 125 ng (2.5 ul loaded) and can be seen on gel

Preparetion

• 50X TAE (Tris-Acetate) Buffer
  121 g/0.5 L Tris
  28.6 g/0.5 L Glacial Acetic Acid
  7.31 g/0.5 L EDTA
  pH 8.0
  
• Sterile 1.5 mL eppendorf tubes
• Sterile long pipette tips
• DNA standards
  • [Aysu]: Promega 1.0 kb (1.0-10Kbp), 100 bp (0.1-1.0 Kbp)
• TAE Buffer
• EtBr sln (10 mg/mL). Purchase as solution ! Store at RT. Light-sensitive and carcinogenic !
• Sample loading buffer. Store at 4 C.

Protocol

• Prepare 360 ml, 1X TAE running buffer




• Prepare gel compartment with tape (do not use parafilm)
• Prepare 60 ml, 1% agarose gel (0.6 g agarose in 60 ml 1X TAE buffer)
  • 1-2 min in microwave. Check and swirl after each 30 sec. It must be dissolved completely.
• Add 1.0 ul EtBr (10 mg/ml) per 10 ml gel solution, swirl to mix
• Add add 4 ul EtBr (10 mg/ml) per 100 ml running buffer.
• Pour gel into gel compartment. Put comb.
• After gel solidifies (30 min), take out the comb and load buffer to one well to measure its capacity.
• Position the gel compartment and fill gel chamber with running buffer.
• Prepare samples and standards (ladder)
• Prepare samples by mixing 4 ul 6X Loading Buffer + 20 ul sample
• Load the samples (for 24 ul sample, set pipette 24 ul).

Do not push the tip to the bottom of the wells. Make a 45 degree angle and support the pipette tip on the edge of the well. Don’t worry, gel will not brake when you take support. Slowly release the sample. Do not use second pipet push not to risk air injection.

• Make the connections: Black (-) on sample side, Red (+) opposite side
• Run at 60V
  5-10V/cm (distance between anode and cathode) is recommended.
  90V causes high heat generation for our unit. Use 60V maximum.
  For overnight runs: 2V is too low, causes diffusion
  35V / 4hrs / ¾ progress
• Gel can be stored in 4 C until next day after completion

Gel Photo

• Adjust zoom and position using visible light







• Before turning on UV load your settings file which has the following parameters:
  • Preview tab, all three options checked
  • Active image
  • Dynamic integration, auto exposure, 10 frames
  • 50/50 brigthness/contrast
  • 0/0 hue/saturation
• Maximize brigthness with camera knob (counterclockwise)
• Turn on UV light
• Lower brightness from camera knob if necessary

 

7. Gel extraction (For Fermentas Gel Extraction Kit)
   
   
   1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut as close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 ml tube and weigh. Record the weight of the gel slice.

   Note: If the purified fragment will be used for cloning reactions, avoid damaging the DNA through UV light exposure. Minimize UV exposure to a few seconds or keep the gel slice on a glass or plastic plate during UV illumination.

   2. Add 1:1 volume of Binding Buffer to the gel slice (volume: weight) (e.g., add 100 µl of Binding Buffer for every 100 mg of agarose gel)

      p>Note. For gels with an agarose content greater than 2%, add 2:1 volumes of Binding Buffer to the gel slice.</p>
   

   3. Incubate the gel mixture at 50-60°C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow
   4. Optional: use this step only when DNA fragment is <500 bp or >10 kb long.
      • If the DNA fragment is <500 bp, add a 1:2 volume of 100% isopropanol to the solubilized gel solution (e.g. 100 µl of isopropanol should be added to 100 mg gel slice solubilized in 100 µl of Binding Buffer). Mix thoroughly
      • - If the DNA fragment is >10 kb, add a 1:2 volume of water to the solubilized gel solution (e.g. 100 µl of water should be added to 100 mg gel slice solubilized in 100 µl of Binding Buffer). Mix thoroughly.
   5. Transfer up to 800 µl of the solubilized gel solution (from step 3 or 4) to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. 

   Note. If the total volume exceeds 800 µl, the solution can be added to the column in stages. After each application, centrifuge the column for 30-60 s and discard the flow-through after each spin. Repeat until the entire volume has been applied to the column membrane. Do not  exceed 1 g of total agarose gel per column.

   6. Optional: use this additional binding step only if the purified DNA will be used for sequencing <p>Add 100 µl of Binding Buffer to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

    
   7. Add 700 µl of Wash Buffer (diluted with ethanol as described on p. 3) to the GeneJET™ purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
   8. Centrifuge the empty GeneJET™ purification column for an additional 1 min to completely remove residual wash buffer.

   Note. This step is essential to avoid residual ethanol in the purified DNA solution. The presence of ethanol in the DNA sample may inhibit downstream enzymatic reactions.

   9. Transfer the  purification column into a clean 1.5 ml microcentrifuge tube (not included). Add 50 µl of Elution Buffer to the center of the purification column membrane. Centrifuge for 1 min.

   Note:

   • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µl does not significantly reduce the DNA yield. However, elution volumes less than 10 µl are not recommended.
   • If DNA fragment is >10 kb, prewarm Elution Buffer to 65°C before applying to column
   • If the elution volume is 10 µl and DNA amount is ≤5 µg, incubate column for 1 min at room temperature before centrifugation
   10. Discard the GeneJET™ purification column and store the purified DNA at -20°C
8. Ligation




This procedure is performed with  NEB BioBrick™ Assembly Kit

1. Ligate the Upstream and Downstream Parts into digested Destination Plasmid.
   • 2 uL Upstream Part Digestion
   • 2 uL Downstream Part Digestion
   • 2 uL Desination Plasmid Digestion
   • 2 uL 10* T4 DNA ligase bufferl
   • 2 uL T4 DNA ligase buffer
   • 11 uL dH2O
2. Incubate at RT for 10 min and then heat inactivate at 80 C for 20 min.
3. Transform 2 uL of the ligation product into 50 uL of competent E.Coli cells ( or other suitable host strain).
4. Select using the antibiotic corresponding to the Destination Plasmid.
   
   
9. Glycerol Stock Preparation
   
   
   1. Centrifuge 5 mL overnight cells at 5000 rpm for 5 min.
   2. Discard 4 mL supernatant, remain 1 mL with pellet.
   3. Add 176 uL 15% glycerol  and resuspend the pellet.
   4. Aliquot 100 uL and immediately freeze at -70 C.
      
      
10. Cell imaging

Checklist Procedure

1. Suspension culture preparation
• Dissolve a single colony in 10 mL LB+Amp
• Incubate 14-15 hrs, shaking at 225 rpm
  
  




2. Scale-up of the suspension culture
• Add 2-3 mL of the suspension culture to 100 mL LB+Amp
• Incubate 5-6 hrs, shaking at 225 rpm
  
  
3. Equalizing OD readings
• Take 10 mL samples from GFP/RFP & NC cultures
• Centrifuge at 10000 rpm for 5 mins
• Discard supernatant
• Resuspend pellet in 500 uL 1X PBS
• Take 250 uL & dilute to 3 mL with 1X PBS
• Read in UV-Vis at 600 nm
• Multiply the OD readings by 12
  
• Dilute remaining 250 uL samples as required to make all samples have the same ODvalue, final volume should also be 250 uL
  
  
4. Fluorescent signal reading
• Set parameters
  [GFP]
• Mode: Emission scan
• Config: Digital
• Slit: 1 turn / 2 nm
• Blaze: 750 nm
• Excitation: 395 nm
• Emission range: 475-600 nm
• Average: 3
• Step size: 5 nm
  [RFP]
• Mode: Emission scan
• Config: Digital
• Slit: 1 turn / 2 nm
• Blaze: 750 nm
• Excitation: 570 nm
• Emission range: 585-650 nm
• Average: 3
• Step size: 5 nm
• Transfer samples to small 150 uL cuvette and scan
5. Data Analysis
• Emission maxima:
  GFP 515 nm
  RFP 605 nm
• Record intensity values at 509 nm for GFP & 606 nm for RFP
• Subtract NC intensity value from GFP/RFP value
• Result is the fluorescent signal of the protein in the bacteria
  
  
11. Protein expression
12. Device Experiments