Difference between revisions of "Team:UMaryland/notebook"

Making LB Broth

Note:
Typical procedure calls for 25 g LB Broth powder per 1L of pure water.
We have 500 mL bottles and we do not want to overfill them so we will do a quarter of that amount.
This also helps in that if one bottle gets contaminated, then only a quarter of the LB broth batch will need to be discarded.

LB, broth, food, making, media

1. Mass 6.25 g of LB powder 4 times
2. Add to 4 bottles
3. Add 250 mL pure water in each
4. Swirl to mostly dissolve
5. Seal cap tightly and then unscrew the cap about two turns
6. Cover with aluminum foil
7. Place autoclave tape on at least one bottle
8. Place bottles in a basket
9. Autoclave for 20 minutes on liquid setting
10. Do not add antibiotics to pure broth. Only add to LB used to plate

Making LB Plates

1. Measure out 25 g of LB broth powder for every one litre of plating material desired. (divide as necessary; we typically do this in quarters)
2. Place in desired container
3. Add 15 g of bacto agar for every litre of plating material desired (divide as necessary)
4. Add 1 L of pure water to container (divide as necessary)
5. Swirl until mostly dissolved
6. Seal cap tightly and then unscrew the cap about two turns
7. Cover caps with aluminum foil
8. Add at least one piece of autoclave tape
9. Autoclave for 20 minutes on liquid setting. Do not set exhaust time. Ensure door is sealed and liquid media is selected
10. Allow to cool to about 50 degrees Celsius. Cool until it is warm to touch. Do not forget to add corresponding antibiotic

Adding Antibiotics

Note:
Chloramphenicol is kept in 1000x stock
Ampicillin is kept in 500x stock

1. Thaw antibiotic
2. Add 250 uL CMP to 250mL LB/agar or 500 uL AMP to 250mL LB/agar

plates, lb, food, making, chlor, chloramphenicol, amp, ampicillin, antibiotic

Restriction Digest and Ligation (3A Assembly)

Protocol modified from this source:
https://static.igem.org/mediawiki/2015/f/f6/STHLM_3A_assembly_protocol.pdf

1. Acquire all necessary information including nanodrop concentrations of any miniprepped samples as well as the concentration of the linearized plasmid backbone. Calculate the volume of miniprepped DNA necessary to total approximately 500 ng. Designate the DNA strands that are to be ligated together as either A or B.
2. Create the three master mixes. One will contain EcoRI and PstI, another will contain EcoRI and SpeI, and the last will contain XbaI and PstI. Combine with water and cutsmart buffer in the ratios directed below. The tables direct for 25 µL to be the final volume. This is enough for 5 digestions. Amounts of CutSmart buffer and water can be adjusted as necessary. Enzyme concentration should only be adjusted in the case of an increase in final volume due to micropipette limitations.
3. Prepare linear backbone for digest by measuring out 4 µL (100 ng) of the backbone of choice and 4 µL of the master mix for the linearized plasmid backbone for each ligation planned for the next step into one PCR tube. Mix well with a pipette. Total volume 8uL.
4. Prepare sample A’s by adding 500 ng worth of DNA to a labeled PCR tube. Supplement with water so that the total volume added is 5 µL. Add 5 µL of master mix A to each tube. Total volume 10 uL.
5. Prepare sample B’s by adding 500 ng worth of DNA to a labeled PCR tube. Supplement with water so that the total volume added is 5 µL. Add 5 µL of master mix B to each tube. Total volume 10 uL.
6. Place all samples in the incubator (set at 37 degrees Celsius) for at least one hour. Longer durations are acceptable. They can be left overnight.
7. Heat kill restriction enzymes at 80 degrees celsius in the the thermocycler (or another apparatus) for 20 minutes.

Ligation

1. Add 2 µL of backbone, 2 µL of enzyme A, and 2 µL of enzyme B to a labeled PCR tube.
2. Add 1 uL T4 DNA ligase buffer Add 2 uL ddH2O
3. Add T4 DNA ligase, total volume 10 uL
4. Allow to sit at room temperature for at least one hour. It can be left overnight at room temperature.

restriction, digest, and, ligation, (3A, Assembly), 3A, Assembly, master, mix, EcoRi, PstI, XbaI, heat, kill, pcr, incubator, thermocycler, dna, buffer, ligase, T4

Transformation

1. Set water bath to 42 C
2. Make sure SOC media is not contaminated. LB is also usable
3. Label test tubes and microcentrifuge tubes
4. Obtain competent cells. KEEP ON ICE
5. Thaw ON ICE until cells are thawed
6. If necessary, create 20 uL aliquots of competent cells in microcentrifuge tubes, keep on ice and return to the -80 as soon as possible
7. Add 2 uL of DNA to 20 uL of competent cells in a microcentrifuge tube
8. Pipette to resuspend, but do so gently so as to not harm the cells
9. Place back on ice for 5 minutes
10. Return any unused cells marking date thawed ON ICE
11. Heat shock at 42 C for 30 - 45s
12. Remove from heat then place in ice for 5 mins
13. Add 200 uL of SOC/rescue media. FLAME BOTTLE AND BOTTLE CAP THOROUGHLY
14. Rescue for one hour in incubator with shaker
15. Plate cells on plates with antibiotics overnight at 37 C

Optional-plate 50uL onto plate with differing antibiotic resistance for control

transformation, water, bath, soc, rescue, media,ice,competent, cells, incubator, antibiotic, plate

Preparing Overnights

Note:
Sterilize test tubes, lids, and micropipette tips before using, and perform procedure close to flame.

1. Gather 5 mL culture tubes and label
2. Ignite bunsen burner
3. Remove plates from incubator (if not already done so)
4. Add 5 mL of LB Broth to test tubes. FLAME BOTTLE AND BOTTLE CAP THOROUGHLY
5. Add of (10 uL) ampicillin or (5 uL) chloramphenicol. (Change amount if not from stock solutions of 500X and 1000X)
6. Use a micropipette to obtain one colony from each plate then release colony in the media
7. Eject pipette tip in the test tube as well and close
8. FLAME CULTURE TUBE AND CAP
9. Place in shaker/incubator overnight

preparing, overnights, plasmid, cloning, amp, ampicillin, chlor, chloramphenicol, shaker, incubator, bunsen, burner, lb, broth, media

Miniprep

1. Obtain overnight cultures
2. Label microcentrifuge tubes
3. Use pipette to transfer 2 x 0.63mL of culture to microcentrifuge tubes, leaving pipette tips inside the respective tubes for future use
4. Try to avoid the pipette tips already inside
5. Centrifuge for 5 minutes at 13000 g
6. Dispose of supernatant, being careful not to lose cells (tap sides gently but do not flick)
7. Repeat steps 3, 4, and 5 until all of the culture has been centrifuged
8. Add 250uL of P1 Buffer and mix with pipette
9. Add 250uL of P2 Buffer
10. You can use the same pipette tips as long as you don’t touch the inside of the pipette
11. Invert at least 4-6 times. This is the important step for DNA
12. Add 350 uL of P3 and invert 4-6 more times
13. Centrifuge for 10 minutes at highest setting (13500 x g; protocol calls for 17900 g, but centrifuge in use has max of 13500 g)
14. Remove all supernatant and place in QIAprep 2.0 spin column via pipette
15. Centrifuge for 30-60 seconds. Discard flow through
16. Wash spin column with 0.5 mL Buffer PB
17. Centrifuge for 30-60 seconds. Discard flow through
18. Wash spin column with 0.75 mL Buffer PE
19. Centrifuge for 30-60 seconds. Discard flow through
20. Centrifuge for one more minute and discard flow through
21. Place QIAprep 2.0 column in a clean 1.5 mL microcentrifuge tube
22. Elute DNA a with 25 uL pure water or Buffer EB
23. Set centrifuge to 7500 x g to keep caps from falling off
24. Centrifuge for one minute after letting sit for one minute. DO NOT DISCARD FLOW THROUGH
25. Repeat steps 20-22
26. Dispose of spin column
27. Take microcentrifuge tube and test for concentration in a nanodrop

miniprep, overnight, plasmid, centrifuge, p1, p2, buffer, p3, pe, pb, spin, column, QIAprep, 2.0

Gibson Assembly with G blocks

Standard

1. Acquire materials including NEB kit, G blocks, and linearized backbone
2. G blocks should be spun down in the centrifuge for 3-5 seconds at 3,000 g to reform pellets that may have been affected by shipping
3. Resuspend G blocks in water. To make each the same concentration of 50 ng/μl, 20 μl of TE Buffer should be added to the pelleted g block fragments. (adjust if starting amount is not 1000 ng)
4. If backbone is in solid form this should be resuspended as well and the resultant concentration should be noted
5. Measure out 10 μl of 2X Gibson Assembly Master Mix into container of choice.
6. Add in 100 ng of plasmid to the tube. About 2-3 times molar excess of g block should then be added. (volume needs to be calculated)
7. Pure water should then be added to reach a total volume of 20 μL
8. Incubate the container at 50 degrees Celsius for one hour
9. The plasmid is now ready for transformation and sequencing

Combining Mdh2, HPS, and PHI Using NEB Gibson Kit

1. Acquire materials including NEB kit, G blocks, and linearized pSB1A3 backbone
2. G blocks should be spun down in the centrifuge for 3-5 seconds at 3,000 g to reform pellets that may have been affected by shipping
3. Resuspend G blocks in water. To make each the same concentration of 50 ng/μl, 20 μl of TE Buffer should be added to the pelleted g block fragments
4. If backbone is in solid form this should be resuspended as well and the resultant concentration should be noted
5. Measure out 10 μl of 2X Gibson Assembly Master Mix into container of choice
6. Add in 100 ng (.07 pmol) of plasmid to the tube. About 2-3 times excess of g blocks should then be added. .2 pmol of both Mdh2 and HPS/PHI will be added. This equates to about .18 μg each or 3.6 μL of each solution. (The two fragments differ only by 70 bp in length. As such the difference in the number of moles is marginal here)
7. Pure water should then be added to reach a total volume of 20 μL. This will have to be adjusted for at the time of the procedure as the volume added in by the plasmid is not known yet
8. Incubate the container at 50 degrees Celsius for one hour
9. The plasmid is now ready for transformation and sequencing

gibson, assembly, with, g, blocks, neb, kit, new, england, bioscience, backbone, pellet, te buffer, mdh2, hps, phi, master, mix

Gel Electrophoresis

Gel Casting

1. Measure out 0.75 g agarose for 1.5% gel, 0.5g for 1% gel
2. Add agarose to 50 mL SYBRsafe in .5x TBE in erlenmeyer flask
3. Microwave in 30 s intervals until agarose is dissolved, making sure to cover flask with paper towel
4. Pour into gel mold, add comb and let cool 20 min
5. Take gel out of mold and put into gel chamber

Gel Electrophoresis

1. Cover gel in 1x LB buffer
2. Add 5 uL of ladder to far left well
3. Add 1 uL dye to 5 uL DNA, then load 5uL into each well
4. Run gel at 150 V for 1 hr, until loading dye is about 75% the way to the end

gel, electrophoresis, casting, agarose, sybrsafe, tbe, chamber, comb,mold, ladder, V, volts, wells, buffer

Gel Purification

Note:
Taken from Zymo Research. Meant for use with ZymoClean kit.
http://www.zymoresearch.com/downloads/dl/file/id/34/d4001i.pdf

1. Excise the DNA fragment¹ from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube
2. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100 µl (mg) of agarose gel slice add 300 µl of ADB)
3. Incubate at 37-55 °C for 5-10 minutes until the gel slice is completely dissolved²
4. For DNA fragments > 8 kb, following the incubation step, add one additional volume (equal to that of the gel slice) of water to the mixture for better DNA recovery (e.g., 100 µl agarose, 300 µl ADB, and 100 µl water)
5. Transfer the melted agarose solution to a Zymo-Spin Column in a Collection Tube
6. Centrifuge for 30-60 seconds. Discard the flow-through³
7. Add 200 µl of DNA Wash Buffer to the column and centrifuge for 30 seconds. Discard the flow-through. Repeat the wash step
8. Add ≥ 6 µl DNA Elution Buffer⁴ or water⁵ directly to the column matrix. Place column into a 1.5 ml tube and centrifuge for 30-60 seconds to elute DNA

¹ The amount of agarose excised from the gel should be as small as possible.
² Do not incubate above 60°C. It is important that the gel slice dissolve completely. This can be facilitated by gentle mixing during the incubation.
³ Remove the flow-through by aspiration. Avoid contamination of the collection tube rim.
⁴ DNA Elution Buffer: 10 mM Tris-HCl, pH 8.5, 0.1 mM EDTA.
⁵ Elution of DNA from the column is dependent on pH and temperature. If water is used, make sure the pH is >6.0. Waiting 1 minute prior to elution may improve the yield of larger (> 6 kb) DNA. For even larger DNA (> 10 kb), the total yield may be improved by eluting the DNA with 60-70 C DNA Elution Buffer

gel, purification, zymo, zymoclean, kit, excise, gel, slice, agarose, adb

DNA Precipitation from Filter Paper

1. To recover the DNA, cut the marked circle area that contains the dried plasmid DNA
2. Using clean forceps, insert the filter paper into a 1.5 ml microcentrifuge tube. Add 100 µl of TE buffer or Ultrapure water, vortex briefly and incubate at room temperature for 5 minutes. Vortex again and briefly centrifuge
3. Add 20 µL of 3M sodium acetate (final conc. 0.3M) and mix well
4. Add 300 µL of cold 100% ethanol from -20 C (final conc. 70% EtOH), mix, and centrifuge on high for 10 min
5. Pour out supernatant and wash pellet with 1 mL 70% EtOH
6. Remove EtOH after washing with pipette
7. Dry under vacuum for 5 min

(revised for Braunschweig’s filter paper, which was too large and the conc of DNA was too high to use the procedure above)

1. To recover the DNA, cut the marked circle area that contains the dried plasmid DNA
2. Using clean forceps, insert the filter paper into a 1.5 ml microcentrifuge tube. Add 750 µl of TE buffer or Ultrapure water, vortex briefly and incubate at room temperature for 5 minutes. Vortex again and briefly centrifuge
3. Split into two 1.5 ml microcentrifuge tubes, leaving filter paper in original
4. Add 75 µL of 3M sodium acetate (final conc. 0.3M) to each tube and mix well
5. Add 1125 µL of cold 100% ethanol from -20 C (final conc. 70% EtOH) to each tube, mix, and centrifuge on high for 10 min
6. Pour out supernatant and wash pellet with 7.5 mL 70% EtOH
7. Remove EtOH after washing with pipette
8. Air dry

dna, precipitation, from, filter, paper, plasmid, sodium acetate, ethanol, etoh, pellet, dried, te, buffer, vacuum

Autoclaving Waste

1. Put biohazard bag in an autoclave tray
2. Put in autoclave in ‘wrapped’ setting for 1 hour heating and 2 hour drying
3. Make sure waste is not liquid and has dried
4. Put the autoclaved waste inside a regular trash bag and make sure the regular trash bag is tied
5. Take waste to the black dumpster behind the building

autoclaving, waste, autoclave, biohazard, trash