03/13/13

Making and Preparing Agarose Gel

1. In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
2. Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
3. Run flask bottom under water to cool agarose
4. Pour into gel rig with comb inserted
5. Let gel cool until opaque

Running Gel of Post-Digested pSB1C3 and pSB1A3

1. Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
2. Remove comb slowly
3. Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
5. Load samples into wells after 1 kb DNA ladder is loaded into far left lane
• well 1:Ladder
• well 2:pSB1C3 EcoR1
• well 3:pSB1C3 EcoR1 Pst1
• well 4:pSB1C3 Pst1
• well 5:pSB1A3 EcoR1
• well 6:pSB1A3 EcoR1 Pst1
• well 7:pSB1A3 Pst1
• well 8:pSB1C3 UNDIGESTED
6. Run gel for 45 minutes-1.5 hours at 85 volts

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03/12/13

Preformed the following restriction digest of pSB1A3 and pSB1C3.

500ng DNA in 20μL

pSB1A3 [DNA] = 135.8ng/μL

pSB1C3 [DNA] = 74.7ng/μL

500ng x μL/135.8μg = 3.68μL

500ng x μL/74.7μg = 6.69μL

Sample	EcoRI	PstI	10X Buffer	DNA	PCR Water	TOTAL
pSB1A3	2μL	0μL	2μL	4μL	12μL	20μL
pSB1A3	2μL	2μL	2μL	4μL	10μL	20μL
pSB1C3	2μL	0μL	2μL	7μL	9μL	20μL
pSB1C3	2μL	2μL	2μL	7μL	7μL	20μL

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03/8/13

Protocol

1. Open Nanodrop 7000 V.6.0
2. Select Nucleic Acid
3. Blank via 3μL of PCR water
4. Verify 0ng/&#;L DNA in PCR water
5. Load 3μL of sample
6. Record DNA concentration in ng/μL
7. Print Screen

pSB1K3

p34KM

pIKM1

pSB1A3

pSB1C3

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02/13/13

Colonies were counted

1:1000 dilution of Ampicillin^R pSB1A3 wasn't properly plated

Dilution	Plasmid	Colony Count	Cells/μg DNA
1:1000	pSB1C3	58	8.24e6
1:1000	pSB1K3	79	1.12e7
1:1000	p34KM	157	2.22e7
1:100	pSB1A3	1521	2.16e7

Note: 1:100 estimates by counting colonies in 1/3 of plate and multiplying by 3

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02/12/13

Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight

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02/13/13

pSB1C3

Dilution 1:1000

58 Colonies

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02/11/13

Transforming Competent Cells

We transformed TOP10 chemically competent cells using plasmids.

Resistance	Plasmid ID	Original Concentration (ng/μL)
Kanamycin	pSB1K3	62
Kanamycin	p34KM	40
Chloramphenicol	pSB1C3	43
Ampicillin	pSB1A3	75

Protocol

1. TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
2. 100ng of plasmid were added to cells
3. Cells were placed on ice for 20 minutes
4. Cells were transformed to a 42°C waterbath for 60 seconds
5. After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
6. Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
7. Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
8. 50mL of each dilution was plated on an LB + Antibiotic plate
9. Plates were incubated at 37°C overnight

Plasmids were diluted to 20μL of 10μg/μL

p34KM

$10ng/\mu L\times \mu L/62ng\times 20\mu L\; =\; 3.2\mu L\; (original\; plasmid\; concentration)\; +\; 16.8\mu L\; (dH$₂O)

pSB1C3

$10ng/\mu L\times \mu L/43ng\times 20\mu L\; =\; 4.65\mu L\; (original\; plasmid\; concentration)\; +\; 15.35\mu L\; (dH$₂O)

pSB1A3

$10ng/\mu L\times \mu L/75ng\times 20\mu L\; =\; 2.6\mu L\; (original\; plasmid\; concentration)\; +\; 17.4\mu L\; (dH$₂O)

pSB1K3

$10ng/\mu L\times \mu L/40ng\times 20\mu L\; =\; 5.0\mu L\; (original\; plasmid\; concentration)\; +\; 15.0\mu L\; (dH$₂O)

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Buffers for Preparing Competent E. coli

02/4/13

TFB1: pH: 5.8/ Sterile Filter

Chemicals	Concentration (mM)
RbCl	100
MnCl2	50
Potassium Acetate	30
CaCl2	10
Glycerol	15% by Weight

TFB2 ph:6.8 (Use KOH to adjust)/ Sterile Filter

Chemicals	Concentrations (mM)
MOPS	10
RbCl	10
CaCl2	75
Glycerol	15% by Weight

TFB1

Chemicals Mass (g) RbCl 3.02965 MnCl2 1.56996 Potassium Acetate 0.74392 CaCl2 0.27806 Glycerol 37.5463	TFB2 Chemicals Mass (g) MOPS 0.53398 RbCl 0.30225 CaCl2 2.88320 Glycerol 37.5457

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Making Antibiotic Stocks

02/1/13

Antibiotics are used to isolate organisms into which plasmids containing antibiotic resistance genes have been transformed.

Make antibiotic plates with the following specs.

Antibiotic	Concentration (µg/mL)	Color Code
Ampicillin	100	Orange
Chloramphenicol	35	Green
Kanamycin	50	Red
Tetracycline	15	Yellow

Stocks should be made at 1000x concentration, so that making 1L of plate medium will require only 1mL of stock antibiotic solution.

Stocks of antibiotics are made at the following concentrations

Ampicillin	100 mg/mL
Chloramphenicol	35 mg/mL
Kanamycin	30 mg/mL
Tetracycline	15 mg/mL

50mL of stock were prepared and split into several 15mL tubes

Stocks should be stored in the refrigerator at 4°C

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01/25/13

Restreaked TOP10 cells on LB plates for isolation of single colonies.

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01/23/13

Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator

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01/18/13

Poured LB agar plates

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01/16/13

Making Agar Plates

LB agar is used to grow our stocks of Escherichia coli

• 10g Bacto Tryptone
• 5g Yeast Extract
• 10g Sodium Chloride (NaCl)
• 15g Agarose

Mix Components in 1L of dH₂O until dissolved. Spilt 1000mL solution into two flask. Cap flask with aluminum to prevent spilling of solution. Autoclave on slow exhaust for 20 minutes. Keep liquid in 65°C water bath to prevent setting until 18-Jan.

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