Estimated time: 3 hours (plus 14-18 hour incubation)

Materials

Procedure

1. Thaw competent cells on ice.
2. Add 50µL of thawed competent cells into pre-chilled 2ml tube.
3. Add 2µL of resuspended DNA to the 2ml tube. Pipet up and down a few times, gently. Keep the competent cells on ice.
4. Close tubes and incubate the cells on ice for 30 minutes.
5. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds.
6. Incubate the cells on ice for 5 minutes.
7. Add 200 μl of SOC or LB media to each transformation
8. Incubate the cells at 37ºC for 2 hours at 260rpm in the shaker.
9. Label petri dishes with the appropriate antibiotic with the part number, plasmid backbone, antibiotic resistance, and date. Plate 200 µl of the transformation onto the labeled antibiotic dishes, and spread. This helps ensure that you will be able to pick out a single colony.
10. Spread remaining transformation onto labeled LB agar plate and spread, approx 50µl.
11. Incubate the plates at 37ºC for 12-18 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivates the antibiotic outside of the bacteria.
12. You can pick a single colony, make a glycerol stock, grow up a cell culture and miniprep.

Controls

Estimated time: 40 minutes

Materials

Procedure

1. Harvest the bacterial culture by centrifugation for 1 min at 10,000 rpm. Pour off the supernatant and blot the inverted tube on a paper towel to remove residue medium. Remove the residue medium completely.
2. Add 250 µL Buffer A1 (Add RNase A to Buffer A1 before use) and completely resuspend bacterial pellet by vortexing or pipetting
3. Add 250 µL Buffer B1, mix gently by inverting the tube 10 times (do not vortex), and incubate at room temperature for 5 minutes.
4. Add 350 µL Buffer N1, mix completely by inverting/shaking the vial for 5 times and sharp hand shaking for 2 times.
   Note: Incubating the lysate in ice for 1 min will improve the yield.
5. Centrifuge the lysate at 13,000 rpm for 10 minutes at room temperature. If the lysate doesn’t appear clean, reverse the tube angle, centrifuge for 5 more minutes and then transfer the clear lysate to DNA column.
6. Carefully transfer the clear lysate into a DNA column with a collection tube, avoid the precipitations, spin at 13,000 rpm for 1 minute, discard the flow-through and put the column back to the collection tube.
7. Add 500 µL Buffer KB into the spin column, centrifuge at 13,000 rpm for 1 minute. Remove the spin column from the tube and discard the flow-through. Put the column back to the collection tube.
8. Add 650 µL DNA Wash Buffer (Add ethanol to DNA wash buffer before use) into the spin column, centrifuge at 13,000 rpm for 1 minute at room temperature. Remove the spin column from the tube and discard the flowthrough.
9. Reinsert the spin column, with the lid open, into the collection tube and centrifuge for 2 minutes at 13,000 rpm.
10. Carefully transfer the spin column into a sterile 1.5 mL tube and add 50-100 µL (> 50 µL) Sterile ddH20 or Elution Buffer into the center of the column and let it stand for 2 minutes. Elute the DNA by centrifugation at 13,000 rpm for 1 minute. Reload the eluate into the column and elute again.
11. Check results on Nanodrop.

Link to NEB Protocol

To determine buffer for double digests

Guide to heat inactivation

General reaction set-up:

Controls

Link to NEB Protocol

1. Make Reaction mixture

Components	20μl Reaction
10X T4 DNA Ligase Buffer*	2μl
Vector DNA (4 kb)	50ng (0.020 pmol)
Insert DNA (1 kb)	37.5ng (0.060 pmol)
Nuclease-free water	to 20μl
T4 DNA Ligase	1μl

2. Ligation temperature and times vary

For inserting a part into backbone (no 3A assembly), the suggested NEB protocol worked	16C overnight or room temperature 10min
For 3A Assembly	Room temperature for an hour, then overnight in 4degree

3. Heat inactivate at 65°C for 10 minutes.


4. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Controls

Reagents and Materials:

Procedures

1. To prepare 0.4% agarose gel for electrophoresis, add 0.4 g of agarose powder into a suitable container with plenty of room to allow the liquid to boil and be swirled.
2. Add 100 ml of 1x TAE electrophoresis buffer and swirl to suspend the powder in the buffer.
3. Place the flask or the bottle into the microwave and place on a medium setting for 3 mins. Stop the microwave every 30 seconds and swirl the flask or bottle to suspend any undissolved agarose. Boil and swirl until all of the agarose gel particles are dissolved.
4. Cool the agarose solution to 55-60ºC. Add 5 µl of 10,000x DuRed and swirl to mix.
5. Prepare the gel casting apparatus and pour the molten agarose into the gel casting tray containing the comb. Allow the agarose to solidify at room temperature for 15-20 minutes.
6. Carefully remove the comb from the solidified gel.
7. Label a microcentrifuge tube for each miniprep sample.
8. DO NOT add loading dye directly to DNA minipreps collection tube. In a separate tube add 1 µl of 6x loading dye and add 5 µl of DNA minipreps sample and pipet up and down to mix.
9. Run the prepared agarose gel under water to saturate the wells. Then place the gel in the electrophoresis chamber and pour electrophoresis buffer into the chamber until it completely covers the gel by 5mm.
10. Load 5 µl of the 1 kb molecular weight ruler into lane one of the gel.
11. Load 6 µl of miniprep sample with loading dye into the wells of the gel.
12. Connect the electrophoresis chamber to the power supply and turn on the power. Make sure that the wells are closest to the black, or negative side.
13. Run the gel at 100V for 30 min, or until the molecular weight ruler is clearly separated. If you need to leave the gel for a longer time (i.e. if you need to go for lunch), you can decrease the voltage to 80V or 90V and run for an hour or 45 minutes, respectively.
    • Note: The voltage and times apply for a 1% agarose gel. For a lower concentration of gel (e.g. 0.7%) it will take less time for the gel to run to completion. We suggest to run the gel at 80V for 30 minutes. For longer times, run the gel at 60V for an hour or at 70V for 45 minutes.
14. Make sure the dye does not run off the gel.
15. Visualize the gel and record the results.

Controls

Estimated time: 45 minutes

Materials
MinElute Midi Gel Extraction Kit

Procedure

1. Pre-weigh a 2.0mL microcentrifuge tube
2. Excise DNA fragment from agarose gel with clean, sharp scalpel. Place in the pre-weighed microcentrifuge tube. Minimize the exposure of the DNA samples under UV and amount of gel cut with DNA.
3. Weigh the tube and calculate the weight of the gel. For every 1 volume of gel (100mg = 100µl) add 3 times the volume of Buffer QG. The maximum amount of gel slice per spin column is 400mg.For >2% agarose gels, add 6 volumes of Buffer QG.
4. Incubate at 50ºC for 10 minutes (or until the gel has completely dissolved). Vortex the tube every 2-3 minutes during incubation to help dissolve the gel. If color is orange or violet after gel slice has dissolved, add 10µl 3M sodium acetate, pH 5.0. The color of the mixture will turn to yellow.
5. Add 1 gel volume of isopropanol to the sample and mix by inverting.
6. Place a MinElute spin column in a provided 2mL collection tube. Apply the sample in the 2.0mL microcentrifuge tube to the MinElute column and centrifuge for 1 min at 13,000 rpm. Discard the flow-through and place the MinElute column back into the same collection tube. If the sample volume exceeds 800µl, simply reload and spin again.
7. Add 500µl Buffer QG to the MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube.
8. Add 750µl Buffer PE to MinElute column and centrifuge for 1 minute at 13,000 rpm. Discard flow-through and place the MinElute column back into the same collection tube. If the DNA will be used for salt-sensitive applications, such as direct sequencing and blunt-ended ligation, let the column stand 2-5 minute after addition of Buffer PE.
9. Centrifuge the column in the 2mL collection tube for 1 minute at 13,000 rpm again to remove residual ethanol.
10. Place each MinElute column into a clean 1.5mL microcentrifuge tube. To elute DNA, add 10µl Buffer EB or water to the center of the MinElute membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.
11. Repeat step 12 but this time load the flow-through back to the membrane. Let the column stand for 1 minute, and centrifuge the column for 1 minute at 13,000rpm.
12. Check Gel extraction product with Nanodrop

Controls

These are the conditions we used to PCR the gBlocks received from IDT. We used Snapgene to design the primers. We used Q5 High-Fideltiy Polymerasefrom New England Biolabs.

Controls

We used TIANquick Mini Purification Kit.

1. Add ethanol (96-100%) to Buffer PW before use (see bottle label for volume).
2. Column equilibration: add 500μl Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB1 back into the collection tube.
3. Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.
4. Transfer the mixture to the Spin Column CB1, incubate at room temperature for 2min. Centrifuge for 30-60s at 12,000rpm. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube.

The maximum loading volume of the column is 800μl. For sample volumes greater than 800 μl simply load again.
5. Add 600 μl Buffer PW (ensure that ethanol has been added) to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm. Discard the flow-through, and place Spin Column CB1 back in the same collection tube.

Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5min after adding Buffer PW, and then centrifuge.
6. Repeat step 4.
7. Centrifuge at 12,000 rpm for 2min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.
8. Place the Spin Column CB1 in a clean 1.5ml microcentrifuge tube. Add 20μl Buffer EB to the center of membrane, incubate for 2min, and centrifuge for 2min at 12,000 rpm