Part of Reserach Log

October 18th
Goal: Start from the beginning by making new ampicillin and plates and checking cell growth.
Methods:
 Prepare 50 mg/mL ampicillin by adding 50 mg of ampicillin power to 1 mL water

 Sterilize it by passing it through a syringe and filter
 Prepare 250 mL new LB broth and 250 mL new –amp and +amp LB agar plates
 Check cell growth by placing 10 microliters of DH5 alpha cells from Iowa City into a 15 mL
polypropylene tube with 10 mL of new media
 In the control tube add only 10 mL of the new media
 Place the two tubes in the shaker incubator overnight
Results:
Discussion: These new items will ensure that everything should be sterile and correct, especially the
ampicillin. The control tube should see no growth and the experimental tube should see cloudy growth.
This cell growth can then be stored in glycerol as a stock solution of DH5 alpha cells.
October 19th
Goal: Check for cell growth in the 15 mL polypropylene tube. If growth is seen, place 150 microliters of
solution on – amp plate and 150 microliters on +amp plate and set in incubator for 12-16 hours.
Hopefully the cells will only grow on the –amp plate. Lastly, make a stock solution of DH5 alpha cells.
Methods:
 From experimental tube place 150 microliters of cells onto a –amp plate
 From the same tube place 150 microliters of cells on to a +amp plate
 Tape shut, label, and place in 37 degrees Celsius incubator for 12-16 hours
 Aliquot 100 microliters of cells and 100 microliters of sterile glycerol into separate centrifuge
tubes
 Place in a new freezer box labeled as stock cells
 Place in -80 degrees Celsius freezer
Results:
Discussion: The growth on both indicates a problem.
October 25th
Goal: Test two types of cells, DH5 alpha and DE3, to see if University of Iowa gave us ampicillin resistant
cells.
Methods:
 Streak on +amp plate DH5 alpha and DE3 stock cells
 Place +amp plate and empty –amp plate in incubator for 12-16 hours
Results:
Discussion: Since no growth was seen, this shows that the cells are ampicillin sensitive and shows that
new competent cells need to be made because they much be contaminated in some way.
October 26th
Goals: Make new calcium chloride and LB media to use to make new competent cells since the last ones
seem to have somehow gotten contaminated.
Methods:
 Calcium chloride  1.47 grams Calcium chloride dihydrate and 100 mL of distilled water
 200 mL LB media
 Autoclave on slow for 20 minutes
 Save for the following week for competent cells
Discussion: By having these new solutions, they should be all sterile and ready to use to make
competent cells and this should ensure the procedure is done in a sterile environment.
November 6th
Goal: Start growing the new cells in media for new competent cells.
Methods:
 In tube  10 mL of media and 10 microliters of cells

 Place tube in 37 degrees Celsius shaking incubator overnight
Discussion: Hopefully this overnight growth will allow for quicker competent cell growth the following
day.
November 7th
Goal: Make new competent cells.
Methods:
 Place tube from incubator into 200 mL media

 Place in 37 degrees Celsius shaking incubator until the absorbance is at 0.5 for a
wavelength of 600 nm using spectrophotometer
 Separate ~35 mL of media and cells into six 50 mL cold polypropylene tubes and ice for
10 minutes
 Centrifuge at 4000 RPMs for 10 minutes
 Decant liquid for 1 minute
 Suspend in 10 mL cold CaCl2 in 3 tubes and dump those 3 suspended tubes into the 3
remaining unsuspended tubes
 Decant fluid for 1 minute
 Suspend in 2 mL cold CaCl2
 Aliquot 100 microliters into centrifuge tubes
 Place in liquid nitrogen
 Place in cold labeled box after being filled and then transfer them to -80 degrees Celsius
freezer
Discussion: These will hopefully be competent and non-contaminated cells that can be used in future
transformations.
November 15th
Goal: Transform 370 ng/microliter pET-21a with the new competent cells and test the competent cells
for ampicillin sensitivity.
Methods:
 Get 100 microliters of cells from the -80 degrees Celsius freezer
 Add 10 microliters of diluted 370 ng/microliter DNA
o Dilution=500 microliters water with 7 microliters 370 ng/microliter DNA
 Place in ice for 20 minutes
 Circulating 42 degrees Celsius water bath for 45 seconds
 Back on ice 1-2 minutes
 Add 800 microliters of LB media
 In dry bath at 37 degrees Celsius until the circulating bath gets back to 37 degrees Celsius, then
back to circulating bath, total of 45 minutes inverting every 5 minutes
 Place 150 microliters on a +amp plate
 Label, invert, tape shut, and place in 37 degrees Celsius incubator for 12-16 hours
Results:
Discussion: Since the cells grew on the – amp plate but not the +amp plate the cells are known to be
alive and ampicillin sensitive. Transformation didn’t work however so it’s unsure if the DNA isn’t
ampicillin resistant or if the cells are competent.
November 17th
Goal: Test the DNA with DE3 cells to see if the DNA is ampicillin resistant and working and that the DH5
alpha cells are the problem.
Methods:
 Add 10 microliters of diluted 370 ng/microliter DNA
o Dilution=500 microliters water with 7 microliters 370 ng/microliter DNA
 Place 150 microliters on a +amp plate
 On a +amp and –amp plate place 50 microliters each of DE3 cells
Results:
Discussion: Seeing growth on the –amp plate showed the cells were alive and no growth on the +amp
showed they were ampicillin sensitive. Not seeing growth on the transformation indicates either the
DNA isn’t ampicillin resistant or that the transformation protocol isn’t working as expected.
November 29th
Goal: Test old DE3 and Rosetta cells for ampicillin and kanamycin sensitivity. Test pET-30 DNA with new
Rosetta cells that are supposed to be competent for transformation procedure effectiveness.
Methods:
 Streak old DE3 and Rosetta cells left from the -80 freezer which was off during the summer on a
+amp, +kan, and –anything plates
 Streak new Rosetta cells on +amp, +kan, and –anything plate
 Place all six plates in the incubator for 12-16 hours
 Place 1 microliter 8.5 ng/microliter pET-30 DNA into 100 microliters new Rosetta cells
 Place on ice for 20 minutes
 Place in circulated 42 degrees Celsius water bath for 45 seconds
 Place in ice 1-2 minutes
 Add 800 microliters LB media
 Place in water bath for 45 minutes inverting every 5 minutes
 Place 150 microliters onto +kan plate, 150 microliters on a +amp plate, and 150 microliters on a
–anything plate
 Place in incubator 12-16 hours
Results
Discussion: Since growth was seen on all three old Rosetta cell plates they were known to be bad and
could be thrown out. DE3 cells should have an ampicillin sensitivity so since they grew on the ampicillin
plate they were thrown out. The new Rosetta cells grew only on the –anything plate which was good
meaning they had sensitivity to both ampicillin and kanamycin. When running a transformation using
pET-30 DNA, no growth was seen on the +amp or +kan plate. The +amp was expected as pET-30 DNA
doesn’t have a resistance for kan, but it does for amp so if the transformation had worked it was
expected to see growth on the +kan plate. This concludes that either the DNA being used isn’t
ampicillin/kanamycin resistant or the transformation protocol isn’t working. New competent DH5 alpha
cells are going to be ordered commercially and the pET-21a will be double digested to ensure it’s exactly
what we think it is.
December 7th
Goal: Double digest pET-21a DNA using the enzymes NDE1 and XHO1. Test in a gel to make
sure the DNA being used in transformation is what it’s thought to be.
Methods:
 Get DNA and enzymes from freezer and keep on ice.
 Centrifuge tube has 1 microliters XHO1, 2 microliters buffer, 1 microliter NDE1, 2.7
microliters 370 ng/microliter DNA, and 13.3 microliters water
o DNA amount calculated by taking 1000/concentration of DNA
o Must equal 20 microliters, always use 2 microliters buffer and 1 microliter of each
enzyme
o Water amount is 20 minus buffer, enzyme, and DNA amounts
 Centrifuge tube for a few seconds
 Place in incubator at 37 degrees Celsius for 1 hour
 Move to 65 degree Celsius incubator for 20 minutes to kill enzymes
 Prep a gel
 Use 20 microliters of DNA mixed with 4 microliters 6x DNA loading buffer
 Load in gel, set voltage to 100 Volts for 30 minutes
 Place under UV light to see bands of digested DNA
Results:
Conclusion: This gel needs to be compared to uncut pET-21a plasmid and a ladder to ensure it’s what
we’re expecting it to be.
February 1st
Goal: Make new materials to run transformations with to test the new competent cells which were
bought previously. Test these cells on + and – amp plates to make sure they’re what we expect. Test the
pET-21a uncut DNA on a gel to compare to the gel ran on the cut pET-21a DNA.
Methods:
 Prepare 250 mL of LB media, 100 mL of LB agar for amp plates, 100 mL of LB agar for Kan plates
for Patrick
 Place flasks into the autoclave for 20 minutes on slow
 Pour plates and store in refrigerator for future use
 Prepare a electrophoresis gel
 Place 20 microliters uncut pET-21a DNA mixed with 4 microliters loading buffer into a well
 Place 5 microliters ladder in the well next to it
 Run at 100 volts for 30 minutes
 Analyze under UV light
 Thaw bought competent cells on ice and aliquot into 100 microliters in centrifuge tubes for
easier use
 Using diluted 300 ng/microliter pET-21a DNA, run transformation
 Dilute 300 ng/microliter pET-21a DNA using 295 microliters of water and 5 microliters
of DNA
 Streak a + and – amp plate with the cells
 Add 10 microliters of diluted DNA to centrifuge tube with cells
 Place 50 microliters on a +amp plate (small plates)
Results:
Lane 1 and 3 = uncut pET-21a DNA and the middle lane shows the ladder
Cell growth seen on –amp plate with cells and transformed cells with uncut pET-21a DNA on an amp
plate. Amp plate with just cells and no DNA showed no growth as expected.
Discussion:
From the given results, we can see that the DNA is what we expect it to be and that the transformation
process seems to be working correctly. The next step is to use these competent cells to grow more and
then test cut/ligated DNA to see if ligation also works.
February 8th
Goal: Cut the pET-21a DNA using a restriction endonuclease and then ligate it back together and test the
process using transformation.
Methods:
 Get DNA and enzyme from freezer and keep on ice.

 Centrifuge tube has 1 microliters XHO1, 2 microliters buffer, 3.33 microliters 300
ng/microliter DNA, and 13.67 microliters water
enzyme
 Centrifuge tube for a few seconds
 Place in freezer
 Test single cut DNA concentration using Take 3
 Single cut using XHO1 had concentration of 50 ng/microliter
 In centrifuge tube place 10 microliters XHO1 cut DNA, 2 microliter ligase buffer, 1
microliter ligase, 7 microliters distilled water for 20 microliters total
o DNA addition = 50 ng, split in half so 10 microliters of 25 ng/microliter
 Leave at room temperature for 30 minutes
 Heat shock at 65 degrees Celsius for 10 minutes to kill ligase
 Start the transformation process
 Place 10 microliters from cut and ligated DNA into 100 microliters competent cells (ligated/cut)
 Place on ice 20 minutes
 Circulated 42 degrees Celsius circulating water bath for 45 seconds
 Ice 1-2 minutes
 Add 800 microliters media
 Place in 37 degrees Celsius dry bath until water bath is down to 42 degrees Celsius, incubate 45
minutes inverting tube every 5 minutes
 Place 150 microliters contents on +amp plate
 Invert plate, label, place in 37 degrees Celsius incubator 12-16 hours
Results: Small growth seen.
Discussion: Since small colonies could be seen, it’s thought that the ligation process is working so going
ahead with my protein and DNA will be the next step.
February 15th
Goal: Make competent cells from the bought competent DH5 alpha cells.
Methods:
 Take cloudy tube from incubator and dump cells and media into remaining ~200 mL of
media
 Place in 37 degrees Celsius shaking incubator until the absorbance is at 0.5 for a
wavelength of 600 nm using spectrophotometer
 Separate ~25 mL of media and cells into six 50 mL cold polypropylene tubes and ice for
10 minutes
 Decant liquid for 1 minute
 Suspend in 10 mL cold CaCl2 in 3 tubes and dump those 3 suspended tubes into the 3
remaining unsuspended tubes
 Decant fluid for 1 minute
 Suspend in 2 mL cold CaCl2
 Aliquot 100 microliters into centrifuge tubes
 Place in cold labeled box after being filled and then transfer them to -80 degrees Celsius
freezer
Discussion: Hopefully these cells, when tested using transformation and uncut pET-21a DNA
used previously, will show growth meaning they’re competent and alive. If this is seen to be true,
research can continue to test the ligation process.
Goal: Make new LB media and LB agar plates.
Methods:
 Prepare 500 mL of LB media
 Prepare 150 mL of LB agar for + and – amp plates
 Autoclave on slow for 20 minutes
 Pour plates and place in refrigerator
Goal: Test the new competent cells with uncut pET-21a DNA.
Methods:
 Using diluted 300 ng/microliter pET-21a DNA, run transformation
 Dilute 300 ng/microliter pET-21a DNA using 295 microliters of water and 5 microliters
of DNA
 Streak a + and – amp plate with the cells
 Add 10 microliters of diluted DNA to centrifuge tube with cells
Results: No Growth
Discussion: No growth was seen in the pET-21a DNA transformed cells showing transformation
failed. This meant either the process is the issue or the new cells which were made. Future
testing will involve running a transformation using both the competent cells made and the
competent cells bought to see if it’s the newly made competent cells or the transformation
process.
February 26th
Goal: Test to see if it’s the newly made competent cells that’re making the transformation not
work or the process itself. Also, make new LB media as the previous solution got contaminated.
Methods:
 Get one test tube of 100 microliters of commercial competent cells
 Get a second of 100 microliters of made competent cells
 Place 10 microliters of diluted 300 ng/microliter pET-21a DNA
o Dilute 300 ng/microliter pET-21a DNA using 295 microliters of water and 5
microliters of DNA
 Streak each cells on a + and – amp plate
 Add 10 microliters of diluted DNA to each centrifuge tube with cells
Results:
Discussion: Since growth was seen in the commercial cells but not the competent cells made by
me, I can infer that the competent cell procedure isn’t working correctly. I’ll use the commercial
cells to use my ligated insert for transformation as it should work.
February 27th
Goal: Start by double-cutting pET-21a DNA with XHO1 and NDE1 for placing my insert in
later.
Methods:
 Use 300 ng/microliter pET-21a plasmid made by Abby Kirchner
 Cut plasmid with XHO1 and NDE1
 In tube place 1 microliter XHO1, 1 microliter NDE1, 2 microliters buffer, 3 microliters
DNA, and 13 microliters distilled water
 Centrifuge and place in 37 degrees Celsius for 1 hour
 Transfer to 65 degrees Celsius incubator for 20 minutes to kill enzyme
 Freeze
March 2nd
 Prep a gel
 In 1 well place 20 microliters cut DNA and 4 microliters loading buffer mixed
 Run at 100 volts for 30 minutes
 Place under UV light and cut out DNA bands
 Extract DNA from gel
o Weigh empty centrifuge tube and tube with gel in it, subtract empty tube from gel
tube to get the weight of the gel ( 1.100 - 1.039 = 0.061 g  61 mg)
o Gel equaled 61 mg, add 61 microliters binding buffer to tube
o Incubate at 50-60 degrees Celsius for 10 minutes
 Transfer to purification column, centrifuge, and discard flow through
 Add 100 microliters binding buffer, centrifuge, discard flow through
 Add 700 microliters wash buffer, centrifuge twice, discard flow through each time
 Transfer purification column to centrifuge tube
 Add 50 microliters distilled water, centrifuge, keep liquid
 Obtain concentration from take 3 test to be 4.15 ng/microliter
 Run a special ligation
o Anneal primers by adding 3 microliters water, 1 microliters of each primer, 2
microliters of buffer to a centrifuge tube
o Mix
o Heat at 90 degrees Celsius for 10 minutes and then cool slowly to room
temperature
o Once cooled add 12 microliters DNA and 1 microliter ligase
o Let sit at room temperature for 30 minutes
o Heat kill at 65 degrees Celsius for 10 minutes
 Start transformation
o Get 100 microliters of competent DH5 alpha cells
o Tube 1  100 microliters cells, 10 microliters double digest and ligated DNA
o Ice for 20 minutes
o 42 degrees Celsius water bath for 45 seconds
o Ice for 1-2 minutes
o Add 800 microliters media
o Incubate at 37 degrees Celsius for 45 minutes inverting tubes every 5 minutes
o On two +amp plates place 150 microliters from each tube
o Let sit 12-16 hours in 37 degrees Celsius incubator
Results:
Discussion: Growth was seen signifying that all portions worked and the gene for the
metallohomeodomain was in fact transformed into DH5 alpha cells. Now I can isolate and send
in for sequencing before transforming into expression DE3 cells.
March 14th
Goal: Grow up the cells transformed with double digested and ligated DNA in LB media.
Methods:
 In around 5 mL of LB media place one colony to grow overnight
Discussion: These cells will multiply and then can be used to isolate the DNA.
March 15th
Goal: Isolate the DNA using a mini prep kit, run a Take 3 on the sample, possibly run gel if
concentration is high enough and not too much DNA will be used.
Methods:
 Use the mini prep kit to isolate the DNA
 Run a take 3 to get a concentration of 11 ng/microliter
 Run in centrifuge for 5 hours
 Add 7 microliters to dried DNA and take Take 3 again to get concentration of 60
ng/microliter
Discussion: This concentration was too low to send to Iowa City to get sequenced so need to do a
midi isolation to get a higher DNA concentration.
March 16th
Goal: Isolate DNA at a high enough concentration for sequencing.
Methods:
 Run a Midi prep kit
 Run Take 3 to get the concentration
 Send to Iowa City for sequencing
Results: Sequencing wasn’t seen as recognized for P4 protein or even PDZ
Discussion: Since none of the sequence was recognizable, it’s suggested that there was a
contamination causing the growth after transformation.
April 5th
Goal: See if my product is right by trying to use same enzymes to cut out insert and run on gel.
Methods:
 Use 397.5 ng/microliter pET-21a plasmid with my insert
 In tube 1 place 1 microliter XHO1, 1 microliter NDE1, 2 microliters buffer, 2.5
microliters DNA, 13.5 distilled water
 In tube 2 place 1 microliter XHO1, 2 microliters buffer, and 2.5 microliters DNA, 14.5
distilled water
 In tube 3 place 1 microliter NDE1, 2 microliters buffer, 2.5 microliters DNA, 14.5
distilled water
 Vortex and place in 37 degrees Celsius for 1 hour
 Transfer to 65 degrees Celsius incubator for 20 minutes to kill enzyme
 Run a gel on cut DNA to see drop off
 Prepare gel
 Place ladder in lane 1
 Place all 20 microliters double digested mixed with 4 microliters running buffer in lane 2
 Place all 20 microliters XHO1 digested mixed with 4 microliters running buffer in lane 3
 Place all 20 microliters NDE1 digested mixed with 4 microliters running buffer in lane 4
 Place 20 microliters uncut DNA with 4 microliters running buffer in lane 5
Results:
Discussion: Most DNA degraded or isolated RNA in the first place. Suggests the RNAse in the
midi prep buffer isn’t working. Need to start over getting new vector with my insert.
April 10th
Goal: Double digest plasmid with XHO1 and NDE1 and test on a gel.
Methods
 Get DNA and enzymes from freezer and keep on ice.
 Centrifuge tube has 1 microliters XHO1, 2 microliters buffer, 1 microliter NDE1, 3.3
microliters 300 ng/microliter DNA, and 12.7 microliters water
enzyme
 Vortex tube for a few seconds
 Prep a gel
 Use 5 microliters of DNA mixed with 1 microliters DNA loading buffer
 In the next lane mix 5 microliters uncut DNA with 1 microliter DNA loading buffer
 In final lane place a ladder
 Load in gel, set voltage to 100 Volts for 30 minutes
 Place under UV light to see bands of digested DNA
Results:
Discussion: This gel shows that the double digested piece is smaller showing that the digestion
worked. This vector can be used to ligate in my DNA insert.
April 17th
Goal: Anneal my primers and ligate them into my vector. Make new LB agar plates with and
without ampicillin along with new LB broth.
Methods:
 Run a special ligaiton
o Mix
temperature
o Once cooled add 4 microliters dd DNA and 1 microliter ligase
 Make new LB plates with and without ampicillin
 Make new LB broth
Discussion: This ligated vector with DNA can be used for transformation.
April 24th
Goal: Run a transformation with the new double digested and ligated pET-21a DNA.
Methods:
 Get 2 tubes of 100 microliters of competent DE3 cells
 Test cells by streaking on + and – amp plates
 Tube 1  100 microliters cells, 10 microliters double digest and ligated DNA
 Tube 2  100 microliters cell, 10 microliters dilute uncut pET-21a DNA
 Ice for 20 minutes
 42 degrees Celsius water bath for 45 seconds
 Ice for 1-2 minutes
 Incubate at 37 degrees Celsius for 45 minutes inverting tubes every 5 minutes
 On two +amp plates place 50 microliters from each tube
 On two –amp plates place 50 microliters from each tube
 Let sit 12-16 hours in 37 degrees Celsius incubator
Results:
Discussion: Since growth was seen on all plates, it’s suggested that the ampicillin used was bad
so new ampicillin must be made and the annealing, ligation, and transformation processes must
be redone after new ampicillin plates are made.
April 26th
Goal: Ligate my DNA into the double digested vector. Anneal the primers before doing that.
Make new ampicillin plates.
 Run a special ligaiton
o Mix
temperature
o Once cooled add 4 microliters dd DNA and 1 microliter ligase
Discussion: After testing the DE3 cells on amp and –amp plates, I can run a transformation with
this ligated vector hoping to see growth.
May 2nd
Goal: After seeing that the amp plates were working, run a transformation to try to get the
matalloenzyme produced and growing in expression DE3 bacterial cells.
 Tube 2  100 microliters cell, 10 microliters dilute uncut pET-21a DNA
 On two +amp plates place 50 microliters from each tube
 On two –amp plates place 50 microliters from each tube
Results:
Discussion: Since growth was not seen on +amp plates, it was okay to do transformation. After
transformation, it was seen that growth occurred on the uncut transformed DNA but not the
double digested. This means either the ligase or the annealing process isn’t working correctly.
May 4th
Goal: Test the ligase through single cut and double cut DNA and ligation, then transformation.
Methods:
 Centrifuge tube has 1 microliters XHO1, 2 microliters buffer, 3.33 microliters 300
ng/microliter DNA, and 13.67 microliters water
 Tube 2 has 1 microliter XHO1, 1 microliter NDE1, 2 microliters of buffer, 3.33
microliters 300 ng/microliter DNA, and 12.67 microliters distilled water
 Tube 3 has 1 microliters XHO1, 2 microliters buffer, 3.33 microliters 300 ng/microliter
DNA, and 13.67 microliters water
 Vortex tubes for a few seconds
 In centrifuge tube 1 place 4 microliters XHO1 cut DNA, 2 microliter ligase buffer, 1
microliter ligase, 13 microliters distilled water for 20 microliters total
 In tube 2 place 4 microliters DD DNA, 2 microliters ligase buffer, 1 microliter ligase, and
13 microliters distilled water
 Leave at room temperature for 30 minutes
 Heat shock at 65 degrees Celsius for 10 minutes to kill ligase
Discussion: Use transformation to test if ligation is working correctly.
May 8th
Goal: Test ligated and non-ligated DNA through transformation to show non-ligated doesn’t
grow alone and hopefully that the ligated does grow showing ligation is working correctly.
Methods:
 Tube 2  100 microliters cell, 10 microliters single cut and ligated DNA
 Tube 3  100 microliters cells, 10 microliters single cut NOT LIGATED DNA
 On three +amp plates place 50 microliters from each tube
 On three –amp plates place 50 microliters from each tube
Results:
Discussion: Growth on all plates show that transformation isn’t for sure working as there must be
a contamination or the ampicillin isn’t at a high enough concentration as non-ligated but cut
DNA shouldn’t allow for cell growth
September 6th 2018

Goal: Test the enzymes and the vector by cutting the vector with XHO1, NDE1, BamH1,
EcoRV, and BamHI with EcoRV
Methods:
 Get six sterile centrifuge tubes and label
 Calculate DNA amount by 1000/ng/microliter = 3.33 microliters pET-21a
 Add 2 microliters of buffer to each
 Add 0.5 microliters of the designated enzyme
 Add distilled water to 20 microliters
 Incubate for 1 hour at 37 degrees Celsius
 Prep a gel
 Run 4 microliters 6X loading dye with 20 microliters of DNA – fill rungs as full as
possible
 Run for 45 minutes at 100 Volts
Results:
From ladder up: pET-21a, XHO1 cut, NDE1 cut, EcoRV cut, BamHI cut, EcoRB and BamHI cut
Discussion: The two enzymes I need for my digest showed cutting ability and the pET-21a is
showing the correct bands so we know the DNA is what we think it is and that the enzymes are
alive and working.
September 12, 2018

Goal: Double cut pET-21a, run in a gel, and purify so I know the enzymes both cut and then the
vector is ready for ligation and transformation.
Methods:
 Add 3.33 microliters pET-21a DNA, 0.5 microliter XHO1, 0.5 microliter NDE1, 2
microliters buffer, and 13.67 microliters of distilled water
 Run in 37 degree water bath for an hour
 Run 20 microliters with 4 microliters running dye in gel
 Cut out double digested band and do a gel extraction purification
Discussion: This pET-21a vector can now be used to ligate in the oligos
September – make comp. cells – protocol in green folder
September 26 2018
Goal: transform uncut DNA into cells to see if competency is high in cells.
Methods:
 100 microliters into 2 tubes that have been cooled on ice
 Dilute DNA twice
o 1 microliters DNA to 999 microliters distilled water
o 3.33 microliters into 100 microliters cells – tube 1
o 1 microliter out of dilution and add into 999 microliters of water (1 pg)
o 3.33 microliters into 100 microliters of cells – tube 2
o 0.5 microliter DNA as is into 100 microliters cells – tube 3 (less colonies?)
 Flick 4 times to mix cells and DNA
 Ice 20 minutes
 Heat 42 degrees C for 45 seconds
 Ice 5 minutes
 Add 900 microliters LB – shake for 45 min at 37 Celsius – in flask and into shaker
o 30-45 minutes
 Plates – pre warm
 100 microliters and plate – First Dilution (1 ng of DNA total)
 Spin all three tubes down in centrifuge – not max speed (1500 and 3000) 3-5 minutes
 Remove and throw away almost all of LB
 Gently try to mix cells into LB (flick)
 Plate everything that’s on tubes on plates
 Incubate overnight
 Send picture to Cooper
Results: Just a sheet of bacteria, not single colonies.
Conclusion: Test the cells to see if they’re amp resistant by themselves or if the plates are
defective.
Prepare 50 mg/mL ampicillin by adding 50 mg of ampicillin power to 1 mL water
Goal: Anneal oligos, ligate into double digested vector, and transform ligated vector into competent
pET-21a cells.
Methods:
 Make 75 mL of LB broth to use for transformation

 Place 1 microliter of each oligo with 48 microliters of annealing buffer and put at 90
degrees Celsius for 5 minutes
 Cool slowly to room temperature
 For ligation, in control place no oligos, 5 microliters double digested pET-21a vector, 2
microliters ligase buffer, 1 microliters ligase, and 12 microliters sterile distilled water
 For next variable, 2 microliters oligos, 2 microliters double digested pET-21a vector, 2
 For last variable, 2 microliters oligos, 5 microliters double digested pET-21a vector, 2
 Place at room temperature for 10 minutes
 Split each 20 microliter solution in half and place three tubes of 10 microliters of ligation
solution into the refrigerator
 In other 3 tubes of ligation, place all 10 microliters into three separate tubes of 100
microliters of competent DH5 alpha cells
 Flick 4 times
 Place in 42 degrees Celsius for 45 seconds
 Place on ice 5 minutes
 Add 900 microliters LB broth
 Place in flask and into shaker incubator for 45 minutes
 Place 3 amp plates into incubator to pre-warm
 After 45 minutes, spin down 3 tubes and decant top layer, resuspend cells in leftover LB
 Plate all that’s left in centrifuge tube on 3 pre-warmed amp plates
 Tape shut and place in incubator overnight

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October 18th

 Prepare 50 mg/mL ampicillin by adding 50 mg of ampicillin power to 1 mL water

Discussion: The growth on both indicates a problem.

 In tube  10 mL of media and 10 microliters of cells

Goal: Make new competent cells.

 Place tube from incubator into 200 mL media

 Get DNA and enzyme from freezer and keep on ice.

Results: Small growth seen.

September 6th 2018

September 12, 2018

September – make comp. cells – protocol in green folder

Prepare 50 mg/mL ampicillin by adding 50 mg of ampicillin power to 1 mL water

 Make 75 mL of LB broth to use for transformation

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