Team:MIT/Protocols


Protocols
C. Hutchinsonii Media

This is the media used for C. Hutch and co cultures with E. Coli. The media contains all the salts needed for both bacteria but not the carbon source (filter paper) due to the carbon source not being liquid. Cultures are made by adding the salt solution to a tube containing a piece of filter paper where the tube with the paper in it has been sterilized (usually by autoclave).

C Hutch ALONE in 1 L

Paper: 50 grams per liter of media, which is one 0.5g 9cm No. 1 filter paper disks per 100 mL.

Component Amount
KH2PO4 0.2 g
MgSO4*7H2O 0.5 g
KCl 0.5 g
NaNO3 0.5 g
FeSO4*7H2O 20 mg
K2HPO4 0.8 g
tryptone 1 g

Autoclave 15 min.

E Coli ALONE (M9) in 1 L

  1. Make 5x M9 salts
    • To make M9 Salts aliquot 800ml H2O and add
      • 64g Na2HPO4-7H2O
      • 15g KH2PO4
      • 2.5g NaCl
      • 5.0g NH4Cl
      • Stir until dissolved
      • Adjust to 1000ml with distilled H2O
      • Sterilize by autoclaving
  2. Measure ~700ml of distilled H2O (sterile)
  3. Add 200ml of M9 salts
  4. Add 2ml of 1M MgSO4 (sterile)
  5. Add 20 ml of 20% glucose (or other carbon source)
  6. Add 100ul of 1M CaCl2 (sterile)
  7. Adjust to 1000ml with distilled H2O
Stock Culture Preparation

C Hutch is slow to grow, so experiments will be much faster if large initial concentrations of C Hutch is used. This is achieved by having a continual stock of C Hutch in the incubator which is used to provide cells for experiments

Protocol:

Stripette:

  • 10ml of current stock
  • 90ml of DSM3T media
  • 5g of autoclaved filter paper strips

into a 500ml flask, and place in shaker.

Transformation
  1. Make sure that the incubator (30/37C) and heat block (42C) are ON.
    1. Put water in the wells of the 42°C heat block.
  2. Make sure required antibiotic plates are present.  Make sure you're using the right antibiotic plates for your plasmid's resistance!
    1. Warm plates to 37°C.  Cold plates reduce transformation efficiency by an order of magnitude.
    2. Also warm 500 µl SOC per transformation to room temperature (if it was in the refrigerator.)
  3. Take the DNA out of --20 freezer, let it thaw.
    1. Vortex DNA to mix, then spin down.  Make sure it is completely thawed out!
  4. Make sure that all of the required reagents/DNA etc are present at the site of transformation before you take the cells out of the -80.
  5. Thaw the competent cells on ice for 3-4 min.
    1. You want to add your DNA right as the last bit of cells' ice melts.  Even if it's still a little slushy, that's okay.
  6. Add 1-2 µl of DNA into the comp cells.  Stir with a pipette tip a few times, then put right back on ice.
    1. If you're transforming the result of a reaction (GG, LR, etc) add 1-2 µl of the reaction.  Don't add more: many of these reactions have additives that screws up transformation.
    2. If you're transforming plasmid DNA (from a miniprep), either (a) dilute it out so you add only ~10 ng of DNA, or (b) plate only 10 µl of the outgrowth – else you'll get a lawn!  Super-coiled DNA transforms super-efficiently. As an alternative, transform 1 ul of miniprep DNA and then streak the outgrowth instead of plating with beads.
    3. You can rescue DNA from an empty mini prep tube by gently pipetting the cells into the empty DNA tube, enough DNA will be stuck to the walls.
  7. Incubate the cells on ice for 30-40 min.
  8. Heat shock the cells for EXACTLY 30 sec at 42 C water bath.
  9. Place back on ice for 90 seconds.
  10. Add 250 ul of SOC (37° to RT) medium to each tube (S.O.C is made by dissolving 0.5 ml of 20% glucose in 25 ml of SOB. Make sure that the SOC is clear and not cloudy/ contaminated.)
  11. Shake the tubes at 37 C, 280 rpm for 60 min.
  12. Plate 100 µl for a reaction product, or 10 µl in a 100 µl puddle of water for a supercoiled plasmid.
  13. Incubate plates upside down overnight at 37 C or 16-18h at 30C.
    Can leave the cells in the incubator for up to 18 hours but no more.
Gel Preparation, Running, and Extraction

Preparing the Gel

  1. Check to see if there is a gel waiting in the fridge.
  2. Dissolve UltraPure agarose to a final concentration of 1%(by mass) in TAE buffer in a glass bottle.
  3. Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. ~1 minute/200ml solution
  4. Let sit until cool enough to handle.
  5. Add 10 µl SYBRSafe (1:10000) per 100 ml of the solution and mix well.
  6. Pour 50ml* of solution per small gel tray. The gel trays and combs should be pre-cleaned with water and wiped dry.
    • Note for combs: 15-well combs hold about 6 ul liquid per well, 12-well combs hold about 15 ul per well, 8-well combs hold about 20 ul per well
      • Taping two 8-well comb wells together results in a well that holds up to 100 ul
      • Taping three 8-well comb wells together result in a well that holds up to 200 ul
  7. Use 120 ml per large gel tray. [need to update amounts]
    1. For the small set: small trays hold 20 ml, large trays hold 50 ml
  8. Wait for the gels to solidify. ~15 mins
  9. Label and store at 4C.

*Most of the iGEM gel trays are the small trays that fit 50ml of solution for making the gel.

Running the Gel

When doing gel extraction, it is important to run both an analytical gel (to view under UV) and an extraction gel (from which bands are excised). UV damages DNA, and so we dont want to expose our extracted DNA.

Analytical Gel:

The analytical gel should have between 20 and 100 ng of DNA in each well. It should be an exact copy of the extraction gel with respect to position, voltage, and run time.

Extraction Gel:

This should be the rest of the digestion(s).

The analytical and extraction gels can technically be part of the same physical gel. Make sure to separate with a razor blade before imaging.

Refer to Gel Prep protocol above to determine the amounts of liquid to load for the specific well.

Appropriate Hyperladder to be used for PCR product which is linear. Usually Hyperladder I will be used.

  1. While casting gel, add two sets of lanes; use one set to load an analytical gel. 
  2. Add 2ul gel loading buffer (Orange G 6X; it helps DNA sink into the bottom of the well) to DNA.
  3. Make sure there is enough 1xTAE in the plate holder.
  4. Load 5.0ul of appropriate hyperladder to one of the lanes.
  5. Load appropriate amount of DNA - As much as possible! Usually 15-18ul - (mixed with the buffer) in each well.
  6. Set the timer and voltage to 100V and 25 min.

Analytical Gel Annotation

The following things need to be added to the analytical gel image BEFORE it is posted to the wiki:

  • Label each lane with part number and amount of DNA loaded
  • Label each band with length and proposed identification
  • Include wt% agarose, run time, and voltage

Gel Extraction Protocol using Zymo kit (preferred if available)

  1. Place the extraction gel on the blue light table.
  2. Cut out the appropriate bands. Place into 2mL microtube(s). Try to cut out as small a piece as possible while still getting all the DNA.
  3. Weigh gel slice (tare with empty microtube). Add 3 volumes of ADB buffer per mg of gel (so a 100mg gel gets 300 uL of ADB buffer).
  4. Incubate at 55C for 10 minutes. Make sure that the gel is completely dissolved.
  5. Add dissolved gel solution to Zymo column in collection tube. Max volume is 800 uL at a time.
  6. Spin 14000 rpm for 30 sec.
  7. Discard liquid in collection tube.
  8. Repeat step 5-7 if had more than 800 uL dissolve gel.
  9. Add 200 uL DNA wash buffer.
  10. Spin 14000 rpm 30 seconds.
  11. Discard liquid in collection tube.
  12. Add 200 uL DNA wash buffer
  13. Spin 14000 rpm 1 min.
  14. Discard liquid in collection tube.
  15. Spin 14000 rpm 1 min one more time (dry spin).
  16. Discard collection tube (but not the column).
  17. (Optional: 2nd dry spin into clean collection tube.)
  18. Place column in a clean labeled microtube.
  19. Add 10 uL (min 6 uL for higher DNA concentration) of sterile DDH2O to top of column. Water should be pipetted directly onto center of filter.
  20. Incubate at RT 1 min (or longer).
  21. Spin 1 min at 14000 rpm. Discard the column.
  22. Measure the concentration on the nanodrop. (You may recover the 1uL from the nanodrop if needed.)

Gel Extraction Protocol using QIAquick Gel Extraction Kit:

  1. Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator. 
  2. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV. 
  3. Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue. 
  4. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 400mg per tube
  5. Add 3 volumes (6 volumes if you are afraid of getting a low yield) of Buffer QG to 1 volume of gel (100mg ~ 100ul)
  6. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
  7. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
  8. Add 1 gel volume of isopropanol
  9. Add max of 770ul to QIAquick column and centrifuge for 1 min (max speed, ~13,000rpm, RT)
  10. Run flow-through over column one more time.
  11. After the second time, discard flow-through and place column back in tube.
  12. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
  13. Add 500uL of Buffer QG to column and centrifuge for 1 min (wash).
  14. Wash: add 0.75ml Buffer PE (make sure that the buffer has ethanol added to it) to column.  Let stand for 2-5 minutes and then centrifuge for 1 min
  15. Discard flow-through & centrifuge for 1 min
  16. Place column into clean Eppendorf tube
  17. Add 50ul Buffer EB or water to center of membrane.  Make sure to use warm EB (50C).  (Use 30uL if worried about low concentration.)
  18. Let stand at RT for 4 min
  19. Centrifuge for 1 min
  20. Measure the concentration using the UV spectrophotometer.
Pro Tips
  1. You don't need 2 lanes if you aren't putting your gel under UV light (the blue light and SYBR safe is fine)
  2. You can up the IPA to 1/4 of the total volume
  3. Warm EB (50 mL conical filled w/ water, plop the tube inside, put it in the heat block)
  4. Don't let it stand at room temperature, you can do it at 5 degrees (heat block)

Gel Extraction Protocol using QIAgen MinElute Kit:

  1. Cut the gel to separate analytical and extraction gel; place analytical gel in UV illuminator. 
  2. Look at the gel under low wavelength UV (high wavelengths will denature DNA). Quickly take a polaroid image and shut OFF the UV. 
  3. Cut extraction gel under white light; avoid UV illuminating the extraction gel as this drastically decreases the DNA yield. If necessary, stain with Methyl Blue. 
  4. Place the cut bands in 2ml Eppendorf tubes; Weigh slices; No more than 300mg per tube
  5. Add 3 volumes of Buffer QG to 1 volume of gel (100mg ~ 100ul)
  6. Incubate at 50C for 10min or until gel is dissolved; vortex every 2-3 min
  7. Confirm that color of mixture is yellow (if not, add 10ul of 3M NaAc, pH 5.0)
  8. Add 1 gel volume of isopropanol
  9. Add max of 800ul to MinElute column and centrifuge for 1 min (speed >= 10,000 G, RT)
  10. Discard flow-through and place column back in tube.
  11. If needed, add rest of mixture to same tube (up to additional 770ul), spin, and discard flow-through
  12. Add 500 uL of buffer QG and spin column for 1 min and discard flow-through
  13. Wash: add 0.75ml Buffer PE(make sure that the buffer has ethanol added to it) to column and centrifuge for 1 min
  14. Discard flow-through & centrifuge for 1 min
  15. Place column into clean Eppendorf tube
  16. Add 10ul Buffer EB (10 mM TrisCl,pH 8.5) or water to center of membrane
  17. Let stand at RT for 1 min
  18. Centrifuge for 1 min
  19. Measure the concentration using the UV spectrophotometer.
Golden Gate
50 ng of each piece of DNA being joined

Use nanodrop to find concentration in ng/ul, then divide 50 by that concentration to find the required volume of DNA: Conc: x ng/uL Vol: 50/x uL

NOTE: If GGDonr is too concentrated, dilute it with EB or water. NOTE: Ligase buffer does not like to be freeze-thawed, so use one-time-use aliquots.

x1 uL of DNA1 x2 uL of DNA2

y uL (100ng) Donor 2ul 10X T4 Ligase Buffer 2ul 10X BSA 1ul BsaI (enzyme) HC (high concentration) 1ul T4 Ligase (enzyme) HC (high concentration)


fill to 20uL with SDIH20 (put water in before the buffer and enzymes) ------------- 20ul total

(NOTE: Make sure that Buffer and Enzyme added last, enzyme after buffer)

Take a p20, set it to 10uL and then pipet up and down.


Example: Excel File

ADD
CONC.
VOLUME
ORDER
DNA1 c1 ng/ul x1 = 50/c1 ul (50ng) 2
... ... ... ...
DNAn cn ng/ul xn = 50/cn ul (50ng) 2
GGDonr d ng/ul y = 50/d ul (100ng) 2
10x T4 Ligase Buffer   2ul 3
10x BSA   2ul 3
BsaI (enzyme)   1ul 4
T4 Ligase (enzyme)   1ul 3
H20    20 - (x1 + ... + xn + y) ul 1
       
TOTAL   20ul  

THERMOCYCLER:

(Protocol EBGG)


37C for 5min

Part 1 50X: 37C for 2.5min 4C for 0.5min 16C for 5.5min

Part 2 37C for 10 min 80C for 20 min

4C hold (for 8+ hours)


(Check protocol by looking up the paper or other online GG protocols)

Making Liquid Cultures

MINIPREP:

  • Prepare culture in a 15 mL, round bottom tube.
  • Add 5mL LB using a seriological pipette
  • Add 5uL of 1000x antibiotic (Ampicillin, Kanamycin, etc.)
  • Pick colony using a 10 ul pipette tip on a p2. Eject tip into tube (tip should remain in tube).
  • if growing from another liquid culture, 100 uL should be plenty (replacing the 1 colony). Almost no amount is too small, just ensure that you get cells.
Polymerase Chain Reaction (PCR)

Appending Prefix and Suffix

Things to keep in mind!

  • Annealing temperature of primers (Tm) should be around 60 C and both primers should be within 1-2C of each other
  • Check the secondary structure of the primers before you order them!
    • no individual secondary structures i.e. hairpins
    • no heterostructure with the forward and reverse primers together
    • free energy of primers should be greater than -4 kCal 
    • GC content should be around 50% (40-60% is okay)
  1. Dilute your DNA to the following concentrations:
DNA Concentrations Template 0.1 - 1 ng/ul
Forward Primer 10 uM
Reverse Primer 10 uM
  1. Set up a small box (e.g. empty pipette tip box) with ice and water. Your DNA and polymerase mix will go into this box before going into the the thermocycler in order to limit endonucelase activity. 
  2. Add the following DNA to a labeled 0.6ml PCR tube 
DNA Volume Template 1 uL
Forward Primer 500 nM
Reverse Primer 500 nM
  1. Program the thermocycler as follows
Temperature Time
98 30s
PAUSE
98 5s
Tm 15
72 (15s)x(#kb)
72 5m
4 forever
  1. Wait for thermocycler to heat up 
  2. Add 22.5uL of polymerase mix (Phusion Master Mix) to your DNA. Mix well and spin down. Transfer tubes to ice as soon as possible. 
  3. Once the thermocycler has heated up to the right temperature (it should be paused at 98C), add tubes to thermocycler and resume PCR program. 

Calculating Reaction Conditions

  1. Use idtdna.com or VectorNTI to calculate melting temperatures of primers

without common overhangs (base pairs 30 to end when read 5' to 3').

PRIMER Tm
FW
RV
  1. Phusion elongates at a rate of 1kb (1000bp) per 15s. Look up the length of the 

gene of interest and calculate time of elongation.

  1. You should get your Tm from NEB.

If you get the melting temperature of your primer from Genious, the annealing

temperature will be that number minus 2.

Assembling Reaction

  1. Get 0.6mL PCR tubes (not the strip tubes).
  2. Get primers for gene of interest. Resuspend if necessary.
  3. Thaw Phusion supermix on ice.
  4. Add the following (in order): 


VOLUME REAGENT
22.5uL (for 35 cycles) Phusion Supermix
2uL 5uM Forward Primer
2uL 5uM Reverse Primer
1uL Template DNA(~150ng)

Programming The Thermocycler

Initial Denaturation: 98C for 5min

LOOP: 30-35 cycles

CYCLE: 
Denaturation: 98C for 10s Annealing: calculated temperature (typically 55-65C) for 30s Elongation: 72C for 15s per kb

Final Elongation: 72C for 10min Store: 4C

Resuspending PCR Primer

From UTexas Protocol:

Materials:

  • Buffer
  • Primer
You will first make a 100 uM master stock:
  1. spin down primer tube in case there is some primer stuck in cap
  2. Calculate amount of buffer needed- multiply nmoles of primer by 10
  3. add this many uL of Buffer to dry DNA pellet
  4. vortex until DNA is dissolved
  5. spin down to recover drops from cap and sides of tube.
Restriction Digest

Digestion Protocol

  • 20 uL Total
    • 500-1000 ng DNA (volume depends on concentration determined by nanodrop)
    • 1 uL enzyme (keep on ice, and add last!)
    • 2 uL enzyme buffer (which buffer depends on the specific enzyme)
    • fill rest with water
  • Pipette up and down thoroughly to mix
  • Incubate at 37 degrees for 1-3 hours
  • 4-5 ul loading dye
  • gel + ladder!

Tips:

Also run a non-digested construct as a control on the digestion itself.

Remember to upload gel to the wiki. Documenting your lab work is required (it is part of your lab notebook). Also remember to keep your page well organized. For ease of calculation (and for keeping track of what is where), use the digest template File:Template Digest.xlsx

From NEB:

One unit is defined as the amount of enzyme required to digest 1 µg of λ DNA in 1 hour at 37°C in a total reaction volume of 50 µl.

So check calculate how much DNA you have and use the right amount of enzyme. Or more.

PCR Purification
TBD
Designing and Ordering Primers

For PCR Primers in Geneious:

  • Highlight the gene that you want to amplify
  • Select "Primers" from the top banner and "design new primers" from the drop down tab
  • Make sure you have checked "Forward Primer" and "Reverse Primer"
  • Set task to "precise" if you are doing this for a cloning reaction
  • check "Target Region"
  • Modify characteristics as needed - see checking Tm and other primer design tips in the protocols page
  • Hit OK and pick the best primer generated - hovering your mouse over the primer will give you Tm and GC content
  • Check your PCR reaction coditions, Geneious may not have an accurate model for predicting Tm, check the NEB website.

For Sequencing Primers:

  • You will get some amount of good read from a primer, look this up. Some primers are standard, they normally read from the ends of the insert. Check the backbone to see what sequencing primers are in place. These may be available from the sequencing shop, or they should at least be in the fridge.
  • If your gene is less than whatever length you can sequence it using back bone primers and do not need to design your own
  • If your gene is larger then what can be read by the back bone primers, you will need to sequence part way through with custom primers.
  • Highlight a 50-100 bp region that you want the primer to bind in
  • Select "Primers" from the top banner and "design new primers" from the drop down tab
  • Set task to "Generic"
  • Check forward primer and DO NOT check reverse primer
  • Check "Included Region"
  • Set "Tm Min" to 50 optimal to 55 and "Tm Max" to 60
  • Set "GC Min" to 45 optimal to 50 and "GC Max to 55
  • Set "GC Clamp" to 1
  • Hit OK and pick the best primer generated - hovering your mouse over the primer will give you Tm and GC content

General tips for sequencing:

Reads are only clear about 30 bp after the primer to 600 bp after the primer.

The easiest way to pick primers is to start with one in a 50 bp region in front of the gene and then measure out 500 bp from that primer then place the next one in the following 100 bp region then measure out 500 bp and place one in the next 100 bp region and so on.

If you are planning on doing additional cloning with this part try to make some of the primers usable for future constructs

For Both:


  • Once your primers are designed. right click on the primer annotation and select "Extract Regions" this will make a primer file in the same folder
  • Select all of your primers and then click File => Export
  • Export the documents as a .csv
  • Highlight only name and sequence
  • Email the csv to your supervisor to be ordered