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After 824: possible growth
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05/22/13

Procedure as preformed on pg. 38

Ligation:

• Insert, Vector: 21μL
• Ligase Buffer: 5μL
• Ligase: 3&#956:L

Let reaction sit on bench for 90 minutes

OR

For overnight ligation: 14°C water bath

05/9/13

We’re repeating this procedure, due to that fact that days between a digestion and ligation shows to be ineffective. Hence, a ligation should be completed soon after digestion.

Place in 37°C water bath for 15 minutes

Ran gel

Used wrong kit (Spin mini prep). Suppose to use PCR purification kit.

Cleaned/purified digests

Clostridial Origin

pSB1C3

Ligation:

• 5μL 10x Buffer
• 2μL ligase (quick)
• ?μL vector
• ?μL insert
• Up to 50μL dH₂

[(ng vector X size insert in Kb)/Size vector in Kb] X molar ratio of insert : vector = ng of insert

Since we used the wrong kit, we’re going to precipitate the DNA out (used digestion on May/9)

Phenol Chloroform Protocol pH 8:

• Turn on heating block
• Add equal amounts of phenol and whatever volume DNA is in (20μL)
• Phenol : Pipette below top surface
• Vortex
• Centrifuge for 30 seconds
• Two layers appear
• Protein is inactive and at interphase
• Pipette out top layer of both and combine aqueous
• Need to get rid of phenol because it absorbs same wavelength as does water and interferes with enzyme activity
• Add 50μL of CIAA 24:1
• Removes Phenol
• Vortex
• Centrifuge for 30 seconds
• Remove top layer (aqueous)
• Leave a little behind so not to contaminate aqueous phase with bottom layer
• Add 50μL of CIAA 24:1
• Vortex
• Centrifuge for 30 seconds
• Remove top layer (aqueous)
• Leave a little behind so not to contaminate aqueous phase with bottom layer

Now we have 50μL of DNA

• Add 1/10 volume sodium acetate 3M pH 5.2
• Add 7/10 volume (total) of isopropanol
• Vortex
• Centrifuge for 20 minutes at 14,000 rpm
• Dispose of liquid, leaving pellet inside tube
• Add 50μL of 10% ethanol to get rid of isopropanol
• Centrifuge for 30 seconds
• Dispose of liquid
• Use heat block to dry
• 60°C for 15 minutes
• Leave tube open
• Add 10μL of PCR water to re suspend pellet
• Use 2μL to quantify via nandrop
• Add 1μL Ligase (quick)
• Add 1μL 10x Buffer = 10μL reaction
• Let reaction sit at room temperature for 2 hours

05/9/13

pSB1C3 = 2000 base pairs

Costridial Origin = 700 base pairs

Want to digest 500ng of backbone and 1:1 ratio of backbone to insert

pSB1C3 linearized backbone = 202.1 ng/μL

500ng of pSB1C3: $500ng\; x\; \mu L/202ng\; =\; 2.5\mu L$

1:1 mole ratio of backbone to insert

175ng of insert:

Place in 37°C water bath for 15 minutes

05/9/13

Used QIAquick PCR Cleanup Kit and processed pSB1K3 and pSB1A3 linearized plasmid backbone PCR products

Nanodrop Quantification

05/9/13

05/8/13

Dilute plasmid backbones to 10ng/μL

pSB1K3

pSB1A3

Thermo Cycler (x38)

• 95°C for 2 minutes
• 95°C for 30 seconds
• 55°C for 30 seconds
• 68°C for 3 minutes
• 69°C for 10 minutes

02/12/13

stuff stuff stuff stuff

05/1/13

To have eight reaction vials with 50μL supermix each. Extra is made for potential pipetting error.

pSB1K3

pSB1A3

For our eight reactions, multiply all values (except supermix) above by 10 and add/mix into 250μL of supermix

Alliquot 55μL of supermix , primers, and template into each tube

To prepare dNTP mix

• 10μL of each NYP
• 60μL of dH₂O

PCR for linearized backbone (actual)

To dilute pSB1A3:(135.8 ng/μL)

1μL Template

4μL PCR dH₂O

We didn’t get what we were trying to amplify

04/28/13

Preformed as on pg. 15 with the following modifactions

• Dilutions
  • 1:10
  • 1:100
  • 1:1000

1. 95°C for 45 seconds
2. 52°C for 45 seconds
3. 72°C for 2 minutes

Repeat 30x

32ng/μL of DNA template (pIKM1) successfully produced the Clostridial origin

PCR product was digested using QIAquick PCR purification kit

Note: If solution turns pick, the pH needs to be adjusted with 5μL of sodium acetate

04/24/13

For 0.5L

• 500mL dH₂
• 15g TSB mix
• 1g glucose
• Adjust pH to 7.3
• Place sponge stopper in place
• Open silver valve and black valve
• Set degassing station to 20 psi
• Switch to nitrogen and run for 30 seconds to flush oxygen out of head space
• Place largest needle into media and turn on spin located on stir-plate, then set time for 45 minutes
• Disinfect top of bottle with alchol wipe
• Add sterile wipe filter with new needle
• Need steady stream of bubbles
• Add 0.1mL Resasrin (will be purple until autoclaved)
• Post autoclaved
• Pink color signifies media has been exposed to oxygen
• No color in media signifies media is anaerobic
• Oxygen scavenger
• If black precipitant forms after autoclave and before transfer into media bottles, then too much oxygen present.

• Use acid washed vials
• Place stoppers in ice and water to shrink pores
• Flush bottles and vials with nitrogen
• Place 35mL in each using automated pipette
• Angle stopper with needle (gas still within)
• Leave for 10 seconds
• Bring needle out of bottle and push stopper in
• Crimp top
• Add 15mL to vials
• Flush head space
• Put regular needle in and flushing needle in
• Flush for 3 minutes
• Pull both needles out at same times so you don’t put pressure on tubes

OR

• Switch degassing station to vacuum
• Alternate between vacuum and pressure 10 times
• Let it settle back to position before going back to pressure
• Make sure all needles are in black stopper
• Tubes now have 20 psi and poke with needle to vent
• Turn off tank
• Autoclave immediately
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04/19/13

PCR of Clostridial Origin with New Primers


1. 95°C for 45 seconds
2. 52°C for 45 seconds
3. 72°C for 2 minutes

Repeat 30x



• well 1: 1:10 dilution of template
• well 2: 1:100 dilution
• well 3: 1:1000 dilution
• well 4: negative control
-------------------------------------pic---------------------------------------------


The band we get from the gel is too small to be the origin that we want. We think that we have just been given something other than what we thought we had


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04/19/13

Making Bulk Amounts of Linearized Plasmid Backbones



PCR Supermix High Fidelity	9.6mL
Primer SB-prep 2Eb	65μL
Primer SB-prep 3P-1	65μL
DNA Template	100ng




pSB1K3

$100ng\; x\; \mu L/40ng\; =\; 9.8\mu L\; =>\; 2.5\mu L\; +\; 7.5\mu L\; dH$₂O

pSB1A3

₂O


pSB1C3

₂O


High fidelity aliquots of 100μL for PCR




PCR

1. 95°C for 2 minutes
2. 95°C for 30 seconds
3. 55°C for 30 seconds
4. 68°C for 3 minutes
5. 68°C for 10 minutes



PCR Cleanup: Use Quiagen


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04/9/13

Gel of 8 Apr 2013 Digest Purifications




Row 1
Lane	Sample
1	1 Kb Plus Ladder
2	pIKM1 + RSA1 digest
3	pIKM1
4	pAN1 + RSA1 digest
5	pAN1




85 Volts for 1 hour


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04/8/13

Verification of pIKM1/pANi



Because we may have accidently stimulated the plasmid and mislabeled them previously. Since, no digestion of the plasmid pIKM1 from last weeks experiment is consistent with a methylating plasmid, today we are digesting both plasmids and running a gel.




This should verify that either we mixed up our plasmids, or we received the wrong plasmid.




1) Plasmid preparation of pAN1 and pIKM1 via Qiagen QiaPrep Spin Miniprep Unit instructions




2) Nanodrop quantification of plasmid DNA


-----------------------------PIC-----------------------------------------------


3) Digest of pIKM1 and pAN1 with RSA1



pAN1

$500ng;times;\mu L/51ng\; =\; 9.8\mu L\; =>\; 10.0\mu L$

pIKM1





Compnents	pAN1	pIKM1
DNA	10μL	2μL
RSA1	2μL	2μL
10x Buffer	2μL	2μL
PCR Water	6μL	14μL
TOTAL	20μL	20μL




Placed in 42°C water bath for 15 minutes


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04/5/13

Row 1
Lane	Sample
1	1 Kb Plus Ladder
2	pSB1C3 + RSA1 + Xba + Invitrogen Buffer
3	pSB1C3 + RSA1 + Xba + Fermentas Buffer
4	pSB1C3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
5	pSB1A3 + RSA1 + Xba + Invitrogen Buffer
6	pSB1A3 + RSA1 + Xba + Fermentas Buffer
7	pSB1A3 + RSA1 + Xba + Invitrogen Buffer + Fermentas Buffer
8	PIKM1 + RSA1
9	pSB1K3 + Hind1 + Xba
10	Negative Control
11	PIKM1
12	pSB1K3


-------------------------------------------------------------------PIC_________------------------
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04/3/13

Since we couldn’t find information about the Fermentas buffer needed for Xba1 and Hind3, we are going to set up our experiment in such a way that tests to see if using only the buffer from Fermentas, using the buffer from Invitrogen, or using a 1:1 mixture of the two will allow our digestion to occur in reactions where enzymes from different companies are being used.




Tubes will be labeled with the plasmid name, which enzyme used, and which buffer was used. For example



• pSB1C3
• RSA1 and Xba1
• 2μL of Invitrogen buffer
• pSB1C3
• RSA1 and Xba1
• 2μL of Fermentas buffer
• pSB1C3
• RSA1 and Xba1
• 1μL of Fermentas buffer and 1μL of Invitrogen buffer



We incubated digests in 42°C water bath for 15 minutes



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04/1/13

Restriction Digest of Biobrick and PIKM1 with AluI



PPIKM1

• 5μL Plasmid
• 2μL RSA1
• 2μL 10x Buffer
• 11μL PCR Water

Biobrick

• 5μL Plasmid
• 2μL RE1(XbaI)
• 2μL RE2(Hind 3 or RSA1
• 2μL 10x Buffer
• 9μL PCR Water




Use a gel of 2% agarose

• 0.8g of agarose
• 40mL of TAE 1x




Regarding a digest, you typically want to match the company that produced the enzyme you’re using to the same company for the buffer. If they’re not the same, look up components and concentrations.


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03/28/13

This looks to be the same size no matter the temperature. We hypothesize that either our primers are laying down non-specifically or they are oriented in the wrong direction. If it turns out our primers are fine, then we may have a different organis, than expected.


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03/27/13

PCR repeat of Clostridial origin repeat



Preformed as outline on page 15 with the following modifications

• Only a 1:100 dilution was used since our DNA template had a concentration of 164.6 ng/μL, which is a good value to use for genomic DNA.

• A temperature gradient for the annealing step was setup where different columns in thermocycler are a different temperature during the annealing process.



Column	Temperature (μC)
2	50.2μC
3	50.8μC
4	51.7μC
5	52.8μC
6	54.1μC
7	55.4μC
8	56.7μC
9	57.9μC
10	58.8μC
11	59.5μC




To set up temperature gradient on thermocycler

1. Highlight stage of interest (in this case; annealing)
2. Selection "options"
3. Select "Show Gradient"

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03/25/13

Gel of Clostridial Origin PCR Product

PCR analysis was completed March 13, 2013



• well 1:Ladder
• well 2:10 Dilution
• well 3:100 Dilution
• well 4:1000 Dilution
• well 5:10000 Dilution
• well 6:Control



From these gels, we can see a band between 250 and 500 bases, which isn't the size of our clostridial origin. Assuming PCR worked correctly, we should see a band of approximately 1200 base pairs. Since we ran multiple cells with the same result, we are hypothesizing that an error was in the PCR reaction. Therefore, we are going to repeat PCR before we move forward by adjusting the annealing temperature.


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03/15/13

Quantification of PCR Product and Linearized Vector with Chloramphenicol

Nanodrop Protocol

1. Click icon with ND-1000 on computer
2. Click "Nucleic Acids"
3. Wipe off pedestal with chemwipe
4. Load 3°L DI water to initialize and click blank
5. Click "Measure" to verify flat line
6. Load 3°L of sample
7. Click "Measure"
8. Click "Print" screen after sample ID has been typed in

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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

Run gel for 45 minutes-1.5 hours at 85 volts ------------------------PIC PG 15--------------------------------------------------------------------------

PCR Reaction Protocol

1. Combine the following
   • 150μL 2x master mix (polymerase,buffer)
   • 1μL forward primer
   • 1μL reverse primer
   • 148μL DI water (PCR water, UV prior to use)
2. Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
3. In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
4. in fifth tube, only put in step 1 solution as a negative control

Regular PCR Cycle

1. 95°C for 45 seconds
2. 55°C for 1 minute
3. 75°C for 1.5 minutes
4. These three steps are cycled 35 times

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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

Nanodrop DNA Quanitification

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

Recipe: Per 1000 mL

• 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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