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Notebook

September 19, 2012:

DNA Extraction

Data: 215 Cells/5 Squares = 43 cells/square x = 43*100*4*10^(-3) x = 43 cells/square (43/.004 mm^3) 1000100 = 1.075*10^9 cells/mL (2.5*10^(7)) cells / (1.075*10^9 cells) = .023mL of bacteria

Then start the extraction using the mini-prep kit (insert link to mini prep kit protocol )

Turn on the thermomixer when the buffers are being added -Temp at 56 degree C for 10 minutes -Takes around 5 minutes to heat up

Use filter tips since we’re working with DNA

As a funny mess up, we ended up throwing away the glass slip for the hemocytometer!!!

September 26, 2012:

Glycerol stocks + Agar+ Nanodrop

-Prepare 50 % glycerol (25mL H2O and 25 mL glycerin) -Agar w/chloramphenciol a. 100mM PIPES b. 1 M CaCl2 c. 1 L Agar

Nanodrop -add small drop of DiH2O 3 times -dab with kim wipe do not wipe -dab both top and bottom bend -log onto ND1000 program and select nucleic acid -add DIH2O with micropipette and close top 2x - add 1 micro liter of DNA to nanodrop, close and measure -save and print

Results: 24.1 nanogram/microliter 260/280 = 1.82 260/230 = .49 Remember to clean machine with DI H2O after use!

1 M CaCl2 Protocol -calculate morality of 1M CaCl2 Mass = M*Vol*MW Mass = 14.702 grams per 100 mL of H2O Add 60 L H2O to beaker Add 14.702g CaCl2 to beaker and stir Add back to column and fill up to 100mL Use 150 mL filter until

PIPES protocol Weight out 2.307 g of pipes solid Required solution must be at 7pH so add NaOH and measure pH using pH stripes or indicator Once at 7 pH add dH2O until 100mL Use 150 mL filter unit

September 27, 2012:

Antibiotic plates

Calculations (Note: Dilution formula was used) Chloramphenicol: (6mg/mL)x=(.025mg/mL)(250mL) x = 1mL Kanamycin: (25 mg/mL)x = (0.05 mg/mL)(250mL) x = .5mL Streptomycin: (20mg/mL)x = (0.02 mg/mL)(250mL) x = .25 mL

Antibiotic Plates (Con’t): Data 1. Chloramphencial: Recommended = 25microgram/mL, stock = 6 mg/mL 2. Kanamycin: Recommended = 50 microgram/mL, stock = 25 mg/mL 3. Strephtomycin: Recommended = 20 microgram/mL, stock = 20 mg/mL

Note: Color coded the plates: Chloramphenciol (Green) Kanamycin (Orange) Strep/Chloram (Green Black)

Bacterial Incubation with Antibiotics E coli W – no antibiotics FMJ 39- strepromycin (25microgram/mL) JW 228- Kanamycin (25 microgram/mL) Bba_K27300- 1000x Amp

To make 1000x Amp (25mg/mL)V = (25microgram/mL)(5mL) V = 5 microliter

Incubate bacteria in 5mL of LB with antibiotics

Glycerol stocks of bacteria Note: Remember to use Aseptic Technique! -Mix 700 micro liter log phase culture with 300 microliter 50 % glycerol -Vortex -Store into cryotube -place in -80 degree C for storage

Nanodrop for HTCC 2181 -Same procedure as the one above

Results: -260/280: 1.78 -260/230: .17 -2 ng/microliter

260/280: 1.98 260/230: .13 1.4 ng/microliter

260/280: 2.53 260/230: .13 2.9 ng/microliter

TAE buffer 1 x for electrophoresis Calculations C1V1 = C2V2 50V1 = (1x)(1000mL) V1 = 20mL of 50x

Primer Stocks [100 micromolar primered stocks] -AD BB Pfx Add 633 microliter DH20 -AD GSA Bt Add 1213 micro lite rDH2O -FO GSA Tp Add 1314 microliter DH2O FO BB Sfx Add 615 microliter DH2O

Diluted to 10 micromolar working volumes

Incubated FMJ39 and JW 1228 for competent because not sure which strain will be used

September 28, 2012:

PCR & Solution prep

CaCl 2 solution

Need 176 mL = 180 mL so V2 = 180 mL

V1 = C2V2/C1

=(60mM x 180mL)/(1000mM) = 10.8mL (1M) CaCl2

=(15%*180mL)/ (50%) = 54 mL glycerol 50%

= (10mM x 180mL)/(100mM) = 18 mL/82.8mL PIPES (100mM)

=97.2mL/180mL DH2O?

Calculations were done with 200 mL a. 12 mL CaCl2 C1 = .03M Vtot = 200mL b. 60 mL glycerol C1 = .5M c. 20 mL PIPES C1 = .1M

Must be filter sterilized afterwards!

PCR protocol

1. PCR ratios for 2.5x a. H2O 47.6microliter b. MgCl2 1.9 microliter c. 10x buffer 6.3 microliter d. DNTP 1.3 microliter e. Forward Primer 1.3 microliter f. Reverse Primer 1.3 microliter g. DNA template 1 microliter h. Taq Pol .5 microliter

2. Three different MgCl2 concentrations a. 1.5 mM 47.6microliter H2O b. 2.0 mM 47.0 microliter H2O c. 2.5 mM 46.4 microliter H2O

Gel Agarose -Refer to Roche FAQ LabDNA agarose 1) Measured 75 g of agar 2) Ad 50 mL 1xTAE Buffer 3) Pour plate into gel box and let it sit Gel Electrophoresis 1 Load 5 micro liter loading dye into each sample 2. Prepare/plan your well load out

1kB FOl FOl5 FO2 100BP FO25 FO3 FO35 1kb ladder

1kb ADl AD15 AD2 100bp AD25 AD3 AD35 1kD ladder

-Prepare samples -Add buffer to gel and remove combs -lod wells with 10 micro liter of sample and 10 microliter ladder -Run at 60*8D Remove gel/stop running Take image

2) 1KD FO1 FO2 FO3 100kb AD1 AD2 AD 1kD

Using the protocol Professor Garcia Ojeda had produced:

September 29, 2012:

PCR Gel Agarose Images

GE Healthcare: GFx PCR DNA: Gel Band Purification -Able to get DNA out of gel FO: ADH

Eluted with 30 microliter of nuclease free H2O

Introduction of plasmid DNA into cells CaCl2 competency cells -we prepared competent cells today note: step 2 of inoculation to OD540 of 0.375 takes 3.5 hours (estimate) -placed in -70 degree Celsius freezer

October 1, 2012:

Gibson Ligation

Transformation using CaCl2 Trial 2

11:40am Inoculated another 400mL of LB+Strephomyocin with 4 mL of FMJ39 Placed in shaker at 37 degrees Celsius with 250 RPM

1 @ 590nm wavelength (nanodrop)

@ 590nm

Gibson Ligation: Performed Gibson Ligation using Protocol w/kit

Amount of DNA used: ADH: 1.4microliter FO: 1.5 micro liter

Gibson Master Mix: 10 micro liter DH20: 7.1 micro liter Total: 20 micro liter

Gibson fraction gel Ran Gel using results of Gibson ligation step

-Excised marked band and extracted using gel extraction kit -namedropped extracted eluted DNA 14.3 ng/ul, 37.7, 61.1 <-- 3 trial Average 45ng/ul

Setup PCR on Gibson Ligated Parts -leave overnight in PCR machine

Standard Parts Assembly -Set PCR for AD and FO using standard part primers and protocol supplied by Marcos Garcia-Ojeda

-Run and purify on gel

Gel Order (1kb) 1st piece FO1 FO2 Ladder AD1 AD2 2nd piece FO1 FO2 AD1 AD2

-Collect DNA from gel

Nanodrop results

-DNA Digestion of AD and FO Combine in two tubes:

Volume in DNA collection tube

FO: 20uL AD: 50uL

Total DNA taken for restriction enzyme FO: (10uL)(34.3ng/uL) = 343 ng

AD: (10uL)(20.16ng/ul) = 201.6 ng

Ligation Step:

34.3ug/20.7uL = 10ng/x

X = 207uL/34.3

FO = 0.603 uL

201.6ug/20.7uL = 17g/x AD = 1.745 uL

a. RxN Volumes

October 2, 2012:

Transformation

Gel for Gibson-Ligated AD-FO DNA Gel Well Order

1kB Ladder Gel G2 (gene) blank (control)

Excised marked band and extracted using Gel Purification Kit This band should contain the AD FO gene w/Eco R1 and Pstl site

Nanodrop results- 2.7 ng/uL

-PCR for more Gibson Assembly product done by Israel

G1 is DNA G2 is H2O Used 1 uL of DNA from 10/1 @45ng/uL

Transformation -Briefly centrifuged the ligation tubes -place the tubes on ice -thaw the competent cells on ice -add about 50 uL of cells into 4 tubes -one for FO -one for AD -one for straight plasmid -one for negative control -Add 5 uL of ligation rxn into corresponding vial -Tap to mix -Incubate tubes on ice for 30 minutes -Incubate tubes @ 47 degrees for 30s -place tubes back on ice -Incubate the tubes @ 37 degree Celsius, 300 rpm for 1 hr -Spread 20-200uL on plates over night -The on remain cells can be stored @ 4 degree Celsius

Team:UC-Merced/Notebook