Cohesive-end restriction cloning of gBlocks ™ Gene Fragments

Preparing cohesive ends for vector and gBlocks ™ Gene Fragments

1. Add the reaction components to the digestion as in the table below
2. Following digestion, remove 5’ phosphates from vector using an alkaline phosphatase. Do not dephosphorylate the gBlocks Gene Fragment.
3. Following dephosphorylation, gel purify the vector to reduce background and eliminate enzymes, and quantify.
4. Following digestion, purify gBlocks Gene Fragment insert with QIAquick column, and quantify. The gBlock Gene Fragment should not be dephosphorylated.

Ligation:

1. Add the following reaction components in the order listed below, centrifuge briefly, and incubate at room temperature for 5 min.
2. Following digestion, remove 5’ phosphates from vector using an alkaline phosphatase. Do not dephosphorylate the gBlocks Gene Fragment.
3. Following dephosphorylation, gel purify the vector to reduce background and eliminate enzymes, and quantify.
4. Following digestion, purify gBlocks Gene Fragment insert with QIAquick column, and quantify. The gBlock Gene Fragment should not be dephosphorylated.

Product	Amount
Linearized Vector	50 ng
gBlocks Gene Fragment	3-5X molar excess
2X Quick Ligase Buffer	10 ul (each)
Quick DNA Ligase	To 1ul
DNase- and RNase- free water	For a final volume of 20 uL

Preparation of Antibiotic stocks

• Cloramphenicol Stock:

1. Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
2. Dissolve in ethanol and mix gently (It is possible to use Vortex)

• Kanamicin Stock:

1. Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
2. Dissolve in distilled water and mix gently (It is possible to use Vortex).

• Ampicilin Stock:

1. Weight 100 mg of liophylized antibiotic for each ml of stock solution.
2. Dissolve in distilled water and mix gently (It is possible to use Vortex).
3. For each 2 ml of media use 1 ul of stock antibiotic.

Therefore the working concentration for each antibiotic (for use in and Agrobacterium tumefaciens and E. coli) will be:
1. Cloramphenicol: 5ul/ml
2. Kanamicin: 25 ul/ml
3. Ampicilin: 50 ul/ml

CaCl2 Competent Cells

1. Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Work sterile. Grow plate overnight at 37°C.
2. Make sure to autoclave 1 L LB (or your preferred media), 1 L of 100 mM CaCl2, 1 L of 100 mM MgCl2, 100 mL of 85 mM CaCl2, 15% glycerol v/v, 4 centrifuge bottles and caps, lots of microfuge tubes
3. Chill overnight at 4°C 100 mM CaCl2, 100 mM MgCl2, 85 mM CaCl2, 15% glycerol v/v
4. Prepare starter culture of cells
5. Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media – no antibiotics). Grow culture at 37°C in shaker overnight.
6. For the next day, inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker.
7. Measure the OD600 every hour, then every 15-20 minutes when the OD getsabove 0.2.
8. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
9. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g for 15 minutes at 4°C.
10. Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.
11. (Spin #2) Harvest the cells by centrifugation at 2000g in the refrigerated centrifuge (~3000 rpm) for 15 minutes at 4°C.
12. Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
13. (Spin #3) Harvest the cells by centrifugation at 2000g (~3000 rpm) for 15 minutes at 4°C. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.
14. Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85 mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.
15. (Spin #4) Harvest the cells by centrifugation at 1000g (~2100) for 15 minutes at 4°C.
16. Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85 mM CaCl2, 15% glycerol. The final OD600 of the suspended cells should be ~200-250.
17. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.

Heat Shock Transformation of E. coli

Note: Never vortex competent cells. Mix cells by gentle shaking.

1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
   • For an Intact Vector: Add 0.5 ul or less to the chilled cells
   • For a Ligation Product: Add 2-3 ul to the chilled cells.
3. Mix gently by flicking the tube.
4. Chill on ice for 10 minutes. (Optional)
5. Heat shock at 42 °C for 50 seconds.
6. Incubate on ice for 2 minutes.
7. Add 200 ul LB, Terrific or SOC medium and recover the cells by shaking at 37 °C.
8. The recovery time varies with the antibiotic selection.
   • Ampicillin: 15-30 minutes
   • Kanamycin or Spectinomycin: 30-60 minutes
   • Chloramphenicol: 60-120 minutes
9. Plate out the cells on selective LB. Use glass beads to spread the cells. The volume of cells plated depends on what is being transformed.
   • For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
   • For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
10. Incubate at 37 °C. Transformants should appear within 8 – 16 hrs.

Transformation by Electroporation

Preparation of Electrocompetent Cells

Competent cells should never be vortexted, as this will cause them to lyse and release salts into the media. Resuspend cells by pipeting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.

1. The night before the transformation, start an overnight culture of cells. 5 ml LB Amp.
2. The day of the transformation, dilute the cells 100X. 100 ml LB Amp.
3. Grow at 30°C for about 90 minutes.
4. Harvest the cells. When the cells reach an OD600 of between 0.6 and 0.8.
5. Split the culture into 2x 50 ml falcon tubes, on ice.
6. Centrifuge at 4 °C for 10 min at 4000 rpm.
7. Wash and combine the cells.
8. Remove the supernatant.
9. Resuspend the cells in 2x 25 ml of ice cold water.
10. Combine the volumes in a single 50 ml falcon tube.
11. Wash the cells 2 more times.
12. Centrifuge at 4 °C for 10 min at 4000 rpm.
13. Resuspend in 50 ml of ice cold water.
14. Repeat.
15. Wash and concentrate the cells for electroporation.
16. Centrifuge at 4 °C for 10 min at 4000 rpm.
17. Resuspend in 1-2 ml of ice cold water.
18. We will use 200 ul of washed cells per transformation.

Dialysis of PCR or Digestion Products

1. DNA for electroporation must be free of salts to avoid arcing.
2. Float a filter in a Petri dish filled with water.
3. Millipore membrane filter 0.025 uM.
4. Pipet one drop of PCR product onto the filter.
5. 200 ng is needed per transformation.
6. 20 - 100 ul fits well on one filter.
7. Collect the drop after 30 - 45 minutes.
8. The volume will change, but the DNA is not lost.

Plasmid purification by Miniprep

1. Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm in the mini-spin for 1 minute, two times.
2. Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth.
3. Centrifuge the eppendorf tube for 15min at 6000 rpm.
4. Discard the supernatant.
5. Resuspend the pellet in 250ml Resuspension buffer.
6. Add 250ml of Lysis Buffer.
7. Gently mix the tube carefully inverting it 5 times carefully.
8. Add and mix softly 350ml of Precipitation Buffer, inverting the tube.
9. Centrifuge it at 12000 rpm for 10 minutes.
10. Transfer the supernatant into a spin column inside a washtube.
11. Centrifuge it at 12000 rpm for a minute.
12. Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column.
13. Incubate it for one minute at room temperature.
14. Centrifuge the column at 12000 rpm for 1 minute.
15. Discard the liquid from the washtube and place the column inside the tube.
16. Add 700ml of Wash Buffer W9 with ethanol to the column.
17. Centrifuge the column with the washtube at 12000 rpm for 1 minute.
18. Discard the liquid from the washtube.
19. Centrifuge the column with the washtube at 12000 rpm for 1 minute.
20. Discard the liquid from the washtube.
21. Place the column inside an eppendorf tube of 1.5ml.
22. Add 75ml of preheated TE Buffer at the center of the column.
23. (Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes).
24. Incubate the column for 1 minute at room temperature.
25. The column was centrifuged at 12000 rpm for 2 minutes.
26. (The eppendorf tube contains the purified plasmid).

Plasmid purification by Midiprep

1. Harvest the cells by centrifugation for 10 min at 12000 rpm in the mini-spin. Discard the supernatant.
2. Resuspend the pelleted cells in 2 mL of Resuspension Solution auditioned with RNase solution. The bacterial pellet should be resuspended by vortexing or pipetting up and down until no cell clumps remain.
3. Add 2 mL of Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Incubate for 3 min at room temperature.
4. Add 0.5 mL of the Endotoxin Binding Reagent. Mix immediately by inverting the tube 5-8 times.
5. Incubate for 5 min at room temperature. Note. After the addition of the Neutralization Solution and Endotoxin Binding Reagent it is important to mix gently, but thoroughly, to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should appear cloudy and contain white precipitate.
6. Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
7. Centrifuge for 10 min at 4,000-5,000 rpm to pellet cell debris and chromosomal DNA.
8. Transfer the supernatant to a 15 mL tube (not provided) by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
9. Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
10. Transfer part of the sample (~ 5.5 mL) to the supplied column pre-assembled with a collection tube (15 mL). Be careful not to overfill the column. Centrifuge for 3 min 10000 rpm in the mini-spin. Discard the flow-through and place the column back into the same collection tube.
11. Repeat the last step to process any remaining lysate through the purification column.
12. Add 4 mL of Wash Solution I (diluted with isopropanol) to the purification column. Centrifuge for 2 min. at 4000 rpm in a swinging bucket rotor. Discard the flow-through and place the column back into the same collection tube.
13. Add 4 mL of Wash Solution II (diluted with ethanol) to the purification 6 column. Centrifuge for 2 min. at 5000 rpm. Discard the flow-through and place the column back into the same collection tube.
14. Repeat the column wash with Wash Solution II
15. Centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to remove residual wash solution. Discard the collection tube containing the flow-through.
16. Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of the Elution Buffer to the centre of the purification column membrane. Incubate for 2 min at room temperature and centrifuge for 5 min at 5000 rpm to elute plasmid DNA.
17. Discard the purification column. Use the purified plasmid DNA in downstream applications or store DNA at -20°C.

Electrophoresis Agarose Gel

1. For each 100 ml of electrophoresis gel, weight 1 g of agarose.
2. Dissolve in the adequate amount of 1x TAE Buffer.
3. Heat in constant agitation. It could be done in microwave by heating in short intervals and agitating manually.
4. When completely dissolved let the flask cool a little then and 1.2 l of ethidium bromide 5 % p/p for each 50 ml of gel.
5. Agitate until it’s completely dissolved. If not used immediately store at 4 °C.
6. Pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).
7. Place newly poured gel at room temperature for 20-30 minutes, until the gel has completely solidified.
8. Once solidified, remove the comb and place the gel into the electrophoresis unit (gel box).
9. Fill the gel box with 1x TAE buffer.
10. Load GeneRuler 1Kb Plus DNA Ladder 0.1 ug/ml weight one lane of the gel.
11. Carefully load your samples into the additional wells of the gel, carefully mixed with 6X DNA Loading Dye.
12. Run the gel at 50-150V until the dye line is approximately 75-80% of the way down the gel (20-45 min).
13. Use an UV light transilluminator to observe the DNA fragments on the gel.

Restriction Digest

1. Keep all enzymes and buffers used on ice.
2. Thaw NEB Buffer 2 and BSA in room temperature water. Mix by shaking the tubes, and flick/spin them to collect the liquid at the bottom of the tube.
3. Add 250ng of DNA to the appropriately labelled tube. Add distilled water to the tubes for a total volume of 16ul in each tube.
4. Pipet 2.5ul of NEB Buffer 2 to each tube.
5. Pipet 0.5ul of BSA to each tube.
6. In the Part A tube: Add 0.5ul of EcoRI, and 0.5ul of SpeI.
7. In the Part B tube: Add 0.5ul of XbaI, and 0.5ul of PstI.
8. In the pSB1C3 tube: Add 0.5ul of EcoRI, 0.5ul of PstI, and 0.5ul of Dpn1.
9. The total volume in each tube should be approximately 20ul. Mix well by pipetting slowly up and down. Spin the samples briefly to collect all of the mixture to the bottom of the tube.
10. Incubate the restriction digests at 37°C for 30 minutes, then 80°C for 20 minutes. We use a thermal cycler with a heated lid.
11. Use ~2ul of the digest (25ng of DNA) for ligations.

Parts Ligation

1. Sticky-end ligation.
2. Prepare the following reaction mixture:

Linear Vector DNA	20 - 100 ng
Insert DNA	1:1 to 5:1 molar ratio over vector
10X T4 DNA Ligase Buffer	2 µl
T4 DNA Ligase	1 u
Nuclease-free water	To 20 µl
Total volume	20 µl

3. Incubate 10 min at 22°C.
4. Use up to 5 µl of the mixture for transformation of 50 µl of chemically competent cells or 1-2 µl per 50 µl
5. The electrotransformation efficiency may be improved by heat inactivation of T4 DNA ligase at 65°C for 10 min or at 70°C for 5 min
6. The overall number of transformants may be increased by extending the reaction time to 1 hour.
7. If more than 2 u of T4 DNA ligase is used in 20 μl reaction mixture, it is necessary to purify DNA (by spin column or chloroform extraction) before electrotransformation.