Team:OU-Norman OK/Project/Notebook
From 2013.igem.org
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
- 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
- 95°C for 45 seconds
- 52°C for 45 seconds
- 72°C for 2 minutes
- well 1: 1:10 dilution of template
- well 2: 1:100 dilution
- well 3: 1:1000 dilution
- well 4: negative control
- 95°C for 2 minutes
- 95°C for 30 seconds
- 55°C for 30 seconds
- 68°C for 3 minutes
- 68°C for 10 minutes
- 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
- 5μL Plasmid
- 2μL RSA1
- 2μL 10x Buffer
- 11μL PCR Water
- 5μL Plasmid
- 2μL RE1(XbaI)
- 2μL RE2(Hind 3 or RSA1
- 2μL 10x Buffer
- 9μL PCR Water
- 0.8g of agarose
- 40mL of TAE 1x
- 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.
- Highlight stage of interest (in this case; annealing)
- Selection "options"
- Select "Show Gradient"
- well 1:Ladder
- well 2:10 Dilution
- well 3:100 Dilution
- well 4:1000 Dilution
- well 5:10000 Dilution
- well 6:Control
- Click icon with ND-1000 on computer
- Click "Nucleic Acids"
- Wipe off pedestal with chemwipe
- Load 3°L DI water to initialize and click blank
- Click "Measure" to verify flat line
- Load 3°L of sample
- Click "Measure"
- Click "Print" screen after sample ID has been typed in
- In a 125mL erlenmyer flask, combine 0.4g ultra pure agarose and 40mL 1x TAE buffer, which makes a 1% gel
- Swirl to mix. Place in microwave for 40 seconds. If all agarose isn't dissolved, heat again in 7 second increments
- Run flask bottom under water to cool agarose
- Pour into gel rig with comb inserted
- Let gel cool until opaque
- Move gel into gel rig container, pour 1x TAE buffer until it covers the gel surface
- Remove comb slowly
- Mix 5-10μL of digest with 3μL of 1:4 EZ Vision dye (Note: if using undigested DNA, only use 2μL
- 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
- Run gel for 45 minutes-1.5 hours at 85 volts
OR
04/19/13
Repeat 30x
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
Back to top04/19/13
PCR Supermix High Fidelity | 9.6mL |
Primer SB-prep 2Eb | 65μL |
Primer SB-prep 3P-1 | 65μL |
DNA Template | 100ng |
pSB1K3
pSB1A3
pSB1C3
High fidelity aliquots of 100μL for PCR
PCR
PCR Cleanup: Use Quiagen
Back to top04/9/13
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
Back to top04/8/13
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
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
Back to top04/5/13
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 |
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
We incubated digests in 42°C water bath for 15 minutes
Back to top04/1/13
PPIKM1
Biobrick
Use a gel of 2% agarose
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.
Back to top03/28/13
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.
Back to top03/27/13
Preformed as outline on page 15 with the following modifications
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
03/25/13
PCR analysis was completed March 13, 2013
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.
Back to top03/15/13
Nanodrop Protocol
03/13/13
Making and Preparing Agarose Gel
Running Gel of Post-Digested pSB1C3 and pSB1A3
PCR Reaction Protocol
- Combine the following
- 150μL 2x master mix (polymerase,buffer)
- 1μL forward primer
- 1μL reverse primer
- 148μL DI water (PCR water, UV prior to use)
- Make 1:10, 1:100, 1:1000, 1:10000 dilutions of template
- In four tubes, combine 50μL of step 1 solution and 1μL of diluted template
- in fifth tube, only put in step 1 solution as a negative control
Regular PCR Cycle
- 95°C for 45 seconds
- 55°C for 1 minute
- 75°C for 1.5 minutes
- These three steps are cycled 35 times
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 |
Back to top
03/8/13
Protocol
- Open Nanodrop 7000 V.6.0
- Select Nucleic Acid
- Blank via 3μL of PCR water
- Verify 0ng/&#;L DNA in PCR water
- Load 3μL of sample
- Record DNA concentration in ng/μL
- Print Screen
pSB1K3
p34KM
pIKM1
pSB1A3
pSB1C3
Back to top02/13/13
Colonies were counted
1:1000 dilution of AmpicillinR 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
Back to top02/12/13
Plates were removed from 37°C incubator, wrapped in parafilm, and stored in 4°C refrigerator overnight
Back to top02/13/13
pSB1C3
Dilution 1:1000
58 Colonies
Back to top02/11/13
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
- TOP10 competent cells in 100μL alliquots (x5) were thawed on ice and resuspended.
- 100ng of plasmid were added to cells
- Cells were placed on ice for 20 minutes
- Cells were transformed to a 42°C waterbath for 60 seconds
- After 60 seconds in waterbath, add 600μL of Psi proth IMMEDIATELY to clls
- Cells were incubated for 60 minutes at 37°C while shaking at 200rpm
- Dilutions of transformation mixture were made at 1:10, 1:100, and 1:1000
- 50mL of each dilution was plated on an LB + Antibiotic plate
- Plates were incubated at 37°C overnight
Plasmids were diluted to 20μL of 10μg/μL
p34KM
pSB1C3
pSB1A3
pSB1K3
Back to topBuffers 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 |
|
|
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
Back to top01/25/13
Restreaked TOP10 cells on LB plates for isolation of single colonies.
Back to top01/23/13
Sealed TOP10 E. coli cells on LB agar plate. Stored in 37°C incubator
Back to top01/18/13
Poured LB agar plates
Back to top01/16/13
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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