Place the SOC medium (from the fridge) in the incubator
Also place X plates in the incubator (With the appropriate antibiotic resistance, Chl for complete, Amp for parts)
Make an ice-water bath
Obtain X MCF tubes and a float
Add 50 ul of competent cells
Add 2ul of the DNA
Place it in the ice-water bath for 2 mins
While waiting, turn on the sink's hot water, obtain a beaker, add hot water, and using a thermometer, adjust water to 42o C (using the hot plate if needed)
Place tubes in the incubator for 30 seconds
Another 2 mins on ice
Add 1 ml of warm SOC medium
Place each tube in the incubator (37o C) for 1 hour
Plating
Pick colonies
Obtain the green sticks, empty LB tubes, and the plates from the incubator
Label the tubes with corresponding plates
Spot the colonies (Choose more than one if available)
Use the sticks and gently obtain the colonies
Swirl around in the LB solution and dispose the stick into the tip trash
Put the tubes into the shaking incubator overnight
Plasmid Prep Procedure
TENS procedure (make fresh each time)-To 4.5 ml TE solution add 250 ul 10% SDS and 250 ul 2M NaOH
Transfer 1.5ml bacterial culture to a labelled MCF tube
Centrifuge for 30 seconds to pellet bacteria
Pour off most of the growth medium. Leave approximately 100ul in the tube
Resuspend the pellet by shaking, vortexing, or tapping the tube vigorously. Make sure the bacteria are completely resuspended and no clumps remain before continuing.
Add 100ul TENS to the tube
Mix well by inversion for 2 minutes. Do not shake the tube. The solution will get viscous
Add 150ul sodium acetate and mix well
Centrifuge for 3 minutes
Transfer the supernatant (liquid) to a clean, labeled MCF tube
Add 1ml 75% Ethanol to the tube and mix well by inversion. You may see DNA as faint white strands in the liquid. The tube should be nearly full of liquid. Sharpie brand pens have an ink that is soluble in Ethanol. Make sure your labels do not get removed accidentally.
Centrifuge 5 minutes to pellet the DNA. Place the tubes in the centrifuge with the hinges out to make finding the pellet easier
Pour off the 95% Ethanol
Place the tubes upside-down and allow them to dry completely
Resuspend the pellet in 25 uL TE
Use 10 uL of the DNA in a restriction digest or 1 uL for PCR
Digest
obtain the plasmid prep tubes (in incubator), new MCF tubes, enzymes in the freezer
For each new tube, prepare the master mix(MM):
water 15ul
buffer 2ul
Upstream enzyme 0.5ul
Downstream enzyme 0.5ul
Each tube needs 18ul of the MM, in order to get enough MM into each tube. If you have x digest, do x+1 MM. (Ex. If I need to do a digest for one tube, I will prepare 30ul of MM, but still put 18ul into the tube)
Label the new tubes with corresponding tubes and change the "P" into a "D"
Add 3ul DNA and 15ul MM into each tube
Obtain a tube float and put all the tubes into the dry incubator for at least an hour.
How to make gel
Obtain one 125ml conical flask, 1x TAE solution, and agarose
With the balance, measure 0.32g of agarose
Use a measuring cylinder and measure 40 ml of 1x TAE solution
Put the measured powder and liquid into the conical flask (make sure no powder is stuck to the wall)
Use plastic film and wrap around the opening of the flask, BUT remember to leave a hole. Do not completely seal the flask
Microwave the solution for 1 minute
Use a glove and transfer the solution from the Microwave to a black top table
Find the well for the gel, duct tape, and combs (depends on the number of digest you have, you need one or two combs)
Tape the edges of the well and make sure the bottom is sealed. Put the combs in
When the microwaved solution is cooled down a bit, add 4 ul of ethidium bromide into the flask and swirl
Pour the solution into the well and wait til it forms a gel like substance
If you need to store it for future uses, obtain a zip lock bag
Tear off the tape carefully, but leave the combs in
Store them into the refrigerator not the freezer
Run the gel
Put 4ul of dyes into each tube of digest, make sure the dye is well mixed
obtain the made gel and put it into the machine (clear one on counter in closet)
Use 0.25x TAE, and pour it until the gel is totally submerged into the solution
Take a paper and record the order of tubes when you put them in
For each roll there are eight empty places **The first place of each roll have to be the ladder (4ul)
Obtain the total 24ul solution from each tube and add into the gel
Close the lid and turn the machine on "high" for approximately 25 minutes
Turn off the machine before removing the lid
Turn off the backroom light and place the gel on the UV light
Turn on the UV light and observe
Ligation
Prepare the mix
Upstream Part digestion - 2ul
Downstream Part digestion - 2ul (replace with water if no Downstream is available, same amount)
Destination Plasmid digestion - 2ul
10X T4 DNA Ligase Buffer - 2ul
T4 DNA Ligase - 1ul
Distilled H2O - 11 ul
Incubate in the incubator for 10 mins
Heat activate at 80oC for 2 mins (hot plate)
Making a Plate
Obtain distilled water, agar, and a 500 ml conical flask (Blue cabinet)
According to the proportion on the label of agar, make a 300 ml solution
Use a magnetic stirring bar, tilt the flask and push the bar into the flask
Put the flask on the hot plate, and turn on the stirring button to high
Seal the opening with aluminum foil and autoclave it with standard procedure
Pull it down and let it cool until solidified
When you need to use it, put it on the hot plate and heat it up for about 10 mins
Then, turn on the stirring button
ALWAYS keep an eye on the flask. When all the solid turns into liquid, stop heating the flask
Transfer the flask to a black surface and when it is warm, add appropriate antibiotics in it (300 ml of agar needs 300 ul antibiotics)
Normally, 300 ml can pour up to 10 plates, so obtain 12 in case
Pour only a thin layer of agar into each plates
Put corresponding color stripes onto the side of the plates
Making different arabinose concentration
Normally we do 0%, 0.001%, 0.01%, 0.1%, 1%, 2%, 4%
The arabinose concentration is in the freezer
Use the LB tubes and add the accurate amount of arabinose in. Remember no matter what the concentration of arabinose is, you cannot add more than 200ul of arabinose solution into the LB. Therefore, if needed switch to use 20% arabinose instead of the 0.2% arabinose with the concentration needs more than 200ul of 0.2% arabinose
Add the cells (from Pick colonies tubes, ask Ms. Byford for amount) into the different concentration
Put them into the shaking incubator overnight
Autoclave
Put everything you want to sterile in the big pressure cooker
Close the lid
Turn the hot plate to 10
Until you hear the "ziiiii" sound, time for 15 mins
When done, put it in the hood and let it cool
Modified Competent Cells
Start liquid culture of DH5 α cells (single colony) from plate. Grow overnight
Get_pre-chilled MCF tubes from fridge
Prepare ice-water bucket
Evenly transfer DH5α cells into MCF tubes
Centrifuge for 10 mins @ 6000 rpm (Use the smaller clinical centrifuge)
Prepare bleach container
Decant supernatant into bleach
Add 400λ of ice cold CCMB80 buffer
Resuspend using the pipet until solution is consistent, then pipet for another 5 cycles (in and out)
Add another 400λ of the buffer. Shake gently by inversion
Place in ice bucket for 20 mins
Centrifuge again for 10 mins. (Same instructions as above)
Decant supernatant into bleach
Resuspend in 175λ of buffer. (Same instructions as above)
Place in ice bucket for 20 mins
Label the float and tubes! (Date and initials!)
Use accordingly or store in large freezer. See "transformation procedure" for more info
Growth Curve Procedure
Procedure (for all figures refer to source 1-a)
Preparation
Materials
E. coli culture(s)
trypticase soy broth
50 ml flask(s)
micropipettes
2 days before experiment
Inoculate a 50 ml flask of trypticase soy broth (TSB) medium with E. coli. Incubate overnight at 27°C. This will
yield 109 CFU/ml.
1 day before experiment
Use 100ml of the prepared culture to inoculate 250 ml of TSB (in a 500 ml flask). Mix thoroughly and remove 5
ml and refrigerate immediately. This is T = 0 and will yield approximately 5 ¥ 105 CFU/ml. Place the flask of E.
coli in a 37°C shaking incubator. Remove 5 ml aliquots of culture every hour up to 8 hours. Store each aliquot
at 4°C. These cultures should be designated T0 through T8.
Procedure
Materials
1 ml aliquots of E. coli broth cultures (or other bacterium)
0.9 ml sterile water dilution tubes in microfuge tubes
micropipettes
poured Petri plates with trypticase soy agar
ice
glass hockey stick spreader
vortex mixer
gas burner
ethyl alcohol for flame sterilization
Make a 10-fold dilution series
For one dilution, transfer 0.1 ml of suspension to each plate. After inoculating all replicate plates in one dilution, go to step 4. Repeat for next two dilutions.
For each plate, sterilize a glass hockey stick spreader in a flame after dipping it in ethanol. Let the spreader cool
briefly. Go to step 5.
Briefly touch the spreader to the agar of an inoculated plate to cool, away from the inoculum. Then, spread the
inoculum by moving the spreader in an arc on the surface of the agar while rotating the plate. Continue until the
inoculum has been absorbed into the agar. Repeat 4 and 5 for the other replicates. Then, go to step 6.
Repeat steps 3, 4, and 5 for each dilution. When done, let the agar dry for a few minutes, tape the plates together,
and incubate them upside down for one week.
Set up a series of dilution tubes to obtain dilutions of 10-1 through 10-7 of the E. coli cultures. Microfuge tubes are
convenient to do this (see Figure 1-3). Each dilution tube will have 900 ml of dilution fluid (sterile saline). A dilution
series will be needed for each E. coli culture (T0 thru T8).
Begin dilutions by adding 100ml of E. coli from the tube labeled T0 which is the initial E. coli culture to tube A.Tube A
is the 10-1 dilution of T0.
Vortex the 10-1 tube for 5 seconds.
Follow this by subsequently adding 100 ml of Tube A to the next tube of saline (Tube B). Tube B is a 10-2 dilution of
T0. Repeat until completing the dilution series, referring to Table 1-2 to see how far you will need to make dilutions
for each E. coli culture. Remember to vortex each tube prior to transfer. It is also important to use a new pipette tip
for each transfer.
Repeat dilutions for T1 through T8 or for whatever samples were assigned to you. Again refer to Table 1-2 to see
how far you need to make your dilutions.
Plate according to the regiment specified in Table 1-2.
Label plates with the dilution and volume to be added to the plate. Make sure the label contains the time point plated
(T1 thru T8) identification. Use triplicate plates for each dilution.
Pipette 100ml from each of the three dilutions to be plated. Add 100ml of each dilution tube to be plated by pipetting
the amount to the center of the agar plate (Figure 1-3).
Immediately spread the aliquot by utilizing a flame sterilized “L” shaped glass rod. If the aliquot is not spread
immediately, it will sorb in situ in the plate resulting in bacterial overgrowth at the spot of initial inoculation.
Repeat the plating for each dilution series for T1 through T8 cultures. Remember to sterilize the rod in between
plates and especially between different dilutions.
Once plates have dried for a few minutes, invert and place in 37°C incubator overnight. Following this, store plates in
refrigerator.
Examining the plates
Examine plates for uniformity of colonies and lack of contamination (see Figure 1-4).
For each culture (T0 through T8), count triplicate plates at one dilution that contains between 30 and 300 colonies.
Calculate the number of cells per ml of original culture for T0 through T8 cultures.
For example, the number of colonies resulting from a 10-4 dilution is 30, 28, and 32.
Mean number of colonies = 30 colonies
These arose from 0.1 ml of a 10^-4 dilution
Number of colonies per ml = (30*10^-4)/0.1
Plot log10 CFU/ml versus time (hours).
From the graph, identify the exponential phase of growth. Using two time points within the exponential phase of
growth and corresponding cell numbers, calculate the mean generation time.
Sources
1. Dilution and Plating of Bacteria and Growth Curves http://booksite.elsevier.com/samplechapters/9780125506564/Sample_Chapter/02~Experiment_1.pdf
