Team:ULaVerne Collab/protocols
GENERAL PROTOCOLS
Transformation Protocol (E. coli)
1. Pre-chill microcentrifuge tube and thaw competent cells on ice.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice for 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8. Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.
2. Add 50uL of competent cells into the chilled microfuge tube.
3. Add 4uL of DNA into the microcentrifuge tube.
4. Incubate on ice for 30 minutes.
5. Heat shock at 42 degrees Celsius for 45 sec.
6. Incubate cells on ice for 2 minutes.
7. Add 200uL of SOC media into the microcentrifuge tube.
8. Incubate and shake at 37 degree Celsius for 1-2 hours.
9. Pipette 200uL of cells into LB plate with ampicillin resistance.
10. Incubate plate overnight at 37 degrees Celsius.
Miniprep
1. Transfer 1.5mL of overnight bacterial cultures from conical tubes to microcentrifuge tubes. Centrifuge at 8000rpm for 3 minutes at room temperature. Discard supernatant and repeat until all of the overnight bacterial culture is suspended.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.
2. Resuspend pelleted bacterial cells in 250uL buffer P1.
3. Add 250uL buffer P2 and mix by inverting 4-6 times.
4. Add 350uL buffer N3 and mix by inverting.
5. Centrifuge for 10 minutes at 1300rpm.
6. Pipette the supernatant from step 5 to the QIAprep spin column. Centrifuge for 1 minute and discard flow through.
7. Wash the QIAprep spin column by adding 750uL buffer PE. Centrifuge for 1 minute and discard flow through.
8. Place the QIAprep column in a clean 1.5mL microcentrifuge tube. Add 50uL nuclease free water to the center of the QIAprep spin column. Let it stand for 1 minute. Centrifuge for 1 minute.
Digestion
1. Thaw all enzymes needed and aliquot appropriate amounts.
2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed)
3. Add appropriate amount of DNA into the nuclease free water.
4. Add 5uL of NEB Buffer to each tube.
5. Add 1uL of the first enzyme to each tube. (BsrG1)
6. Add 1uL of the second enzyme to each tube. (BamHI)
7. Add 1uL of SAP to vector tube.
8. Agitate each solution to mix well.
9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.
10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.
2. Pipette the amount of nuclease free water needed to make 500ug of DNA. (500 ug/Concentration. Then take that amount and subtract it from 50uL to get the amount of water needed)
3. Add appropriate amount of DNA into the nuclease free water.
4. Add 5uL of NEB Buffer to each tube.
5. Add 1uL of the first enzyme to each tube. (BsrG1)
6. Add 1uL of the second enzyme to each tube. (BamHI)
7. Add 1uL of SAP to vector tube.
8. Agitate each solution to mix well.
9. Incubate parts at 37 degrees Celsius for 15 minutes. Incubate vector at 37 degrees Celsius for 30 minutes.
10. Denature the enzymes to end the digestion process by incubating at 80 degrees Celsius for 20 minutes.
Ligation
1. Find the 3:1 (insert:vector) molar ratio of DNA ends. Use NEB ligation calculator.
2. Calculate amount of vector to 50ng vector.
3. Add appropriate amount of nuclease free water into a microcentrifuge tube for a total of volume of 20uL.
4. Add appropriate amount of each parts into the tube with water for a total volume of 20uL.
5. Add 2 uL of 10X T4 DNA Ligase Buffer into the tube.
6. Add 1 uL of T4 DNA Ligase into the tube.
7. Incubate at room temperature for 10 minutes.
8. Heat inactivate at 65 degrees Celsius for 10 minutes.
9. Store in at -20 degrees Celsius freezer or transform ligated DNA.
2. Calculate amount of vector to 50ng vector.
3. Add appropriate amount of nuclease free water into a microcentrifuge tube for a total of volume of 20uL.
4. Add appropriate amount of each parts into the tube with water for a total volume of 20uL.
5. Add 2 uL of 10X T4 DNA Ligase Buffer into the tube.
6. Add 1 uL of T4 DNA Ligase into the tube.
7. Incubate at room temperature for 10 minutes.
8. Heat inactivate at 65 degrees Celsius for 10 minutes.
9. Store in at -20 degrees Celsius freezer or transform ligated DNA.
QIAEX II Gel Extraction Kit Protocol
1. Cut DNA band from the agarose gel with a clean, sharp razor blade. Weigh a 1.7mL centrifuge tube.
2. Add the DNA gel slice to weighed tube. Reweigh and subtract from original tube to find weight of DNA slice.
3. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100bp; 3 volumes for 100bp-4kb; 3 volumes with 2 volumes of water for >4kb; 6 volumes when using >2% agarose gels.
4. Resuspend QIAEX II by vortexing for 30s. Add QIAX II to the tube with the sample and vortex: Use 10µL of QIAEX II for ≤ 2µg DNA; 30µL for 2-10µg DNA; and an additional 30µL for each additional 10µg DNA.
5. Incubate at 50°C for 10min vortexing every 2min to melt agarose and bind DNA. Keep an eye on color change. If so adjust accordingly.
6. Centrifuge the sample for 30s and carefully remove supernatant.
7. Wash the pellet with 500µL Buffer QX1. Resuspend by vortexing. Centrifuge for 30s and remove supernatant.
8. Wash the pellet twice with 500µL Buffer PE. Resuspend pellet by vortexing. Centrifuge for 30s and remove supernatant.
9. Air-dry the pellet until it becomes white. Do not vacuum dry.
10. To elute DNA, add 20µL nuclease free water and resuspend the pellet by vortexing.* Incubate according to fragment size: 5min at room temp. for ≤4kb; 5min at 50°C for 4-10kb; 10min at 50°C for > 10kb.
*Fragments larger than 10kb resuspend by flicking and inverting tube as vortexing can cause shearing.
11. Centrifuge for 30s and pipet supernatant into clean tube. This is your purified DNA.
12. Repeat steps 9 and 10 to combine eluates and increase yield.
2. Add the DNA gel slice to weighed tube. Reweigh and subtract from original tube to find weight of DNA slice.
3. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100bp; 3 volumes for 100bp-4kb; 3 volumes with 2 volumes of water for >4kb; 6 volumes when using >2% agarose gels.
4. Resuspend QIAEX II by vortexing for 30s. Add QIAX II to the tube with the sample and vortex: Use 10µL of QIAEX II for ≤ 2µg DNA; 30µL for 2-10µg DNA; and an additional 30µL for each additional 10µg DNA.
5. Incubate at 50°C for 10min vortexing every 2min to melt agarose and bind DNA. Keep an eye on color change. If so adjust accordingly.
6. Centrifuge the sample for 30s and carefully remove supernatant.
7. Wash the pellet with 500µL Buffer QX1. Resuspend by vortexing. Centrifuge for 30s and remove supernatant.
8. Wash the pellet twice with 500µL Buffer PE. Resuspend pellet by vortexing. Centrifuge for 30s and remove supernatant.
9. Air-dry the pellet until it becomes white. Do not vacuum dry.
10. To elute DNA, add 20µL nuclease free water and resuspend the pellet by vortexing.* Incubate according to fragment size: 5min at room temp. for ≤4kb; 5min at 50°C for 4-10kb; 10min at 50°C for > 10kb.
*Fragments larger than 10kb resuspend by flicking and inverting tube as vortexing can cause shearing.
11. Centrifuge for 30s and pipet supernatant into clean tube. This is your purified DNA.
12. Repeat steps 9 and 10 to combine eluates and increase yield.
QIAquick PCR Purification Kit
1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix.
2. Apply sample to QIAquick purple column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in the same tube.
3. Add 750uL of Buffer PE to column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in same tube.
4. Centrifuge empty column at 13,000 for 1 minute to remove residual wash buffer.
5. Place column in a clean 1.5mL microfuge tube.
6. Add 50uL nuclease-free water to center of membrane. Let column stand for 1 minute and centrifuge for 1 minute.
7. Label microfuge tube containing purified vector.
2. Apply sample to QIAquick purple column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in the same tube.
3. Add 750uL of Buffer PE to column and centrifuge at 13,000rpm for 1 minute. Discard flow through and place column back in same tube.
4. Centrifuge empty column at 13,000 for 1 minute to remove residual wash buffer.
5. Place column in a clean 1.5mL microfuge tube.
6. Add 50uL nuclease-free water to center of membrane. Let column stand for 1 minute and centrifuge for 1 minute.
7. Label microfuge tube containing purified vector.
CHLAMYDOMONAS PROTOCOLS
Transformation Protocol (E. coli)
Culturing and Preserving Chlamydomonas Reinhardtii
Prior to beginning the culturing of the Chlamydomonas
1. Place cells upright away from direct sunlight in a tube rack or beaker. Keep them in a cool area at room temperature until they are ready to be cultured
(DO NOT leave them unattended for more than three days. If they are not going to be treated within the first 36 hours of arrival, transfer the cell culture to a weak nutrient solution like half-strength Alga-Gro® Medium or minimal growth medium).
2. Autoclave anything that will be touching the cell culture (Erlenmeyer flasks, pipette tips, etc.) for 15 minutes at 15 pounds of pressure
3. Set up the (2) 40 watt light bulbs about 45 to 60 cm above the culture
4. Cover the area in foil to retain heat
Beginning the culture
5. In a sterile hood, pipette 200mL of TAP medium into a 500mL Erlenmeyer flask
6. Pipette 10mL of the Chlamydomonas strain into a 500mL Erlenmeyer flask containing the 200mL fresh medium (If the medium is grown in a slant tube, in a sterile field, use an inoculating loop to pull out some of the Chlamydomonas and place it in the 200 mL of fresh medium)
7. Parafilm seal the Erlenmeyer flask to prevent contamination and place the flask on a stir plate on top of the petri dish lid
8. Check it every so often to make sure there is continuous movement (YOU DO NOT WANT ANY SHADOW AREAS)
9. Set a timer to alternate between light and dark every 12 hours
10. Using a spectrophotometer measure the growth of the cell culture everyday at the same times (Set the spectrophotometer at 750nm and use identical Kimax tubes)
11. Incubate the cell culture until the growth curve reaches 0.5
12. Once the cell culture reaches 0.5 on the spectrophotometer and the culture’s cell density of approximately 0.9-2.0 x 107 ml-1 (3-5 days after transfer, late linear phase), aliquot the cells into 1.5mL Eppendorf tubes
Have liquid nitrogen ready in a foam container to snap freeze the aliquots
Prior to beginning the culturing of the Chlamydomonas
1. Place cells upright away from direct sunlight in a tube rack or beaker. Keep them in a cool area at room temperature until they are ready to be cultured
(DO NOT leave them unattended for more than three days. If they are not going to be treated within the first 36 hours of arrival, transfer the cell culture to a weak nutrient solution like half-strength Alga-Gro® Medium or minimal growth medium).
2. Autoclave anything that will be touching the cell culture (Erlenmeyer flasks, pipette tips, etc.) for 15 minutes at 15 pounds of pressure
3. Set up the (2) 40 watt light bulbs about 45 to 60 cm above the culture
4. Cover the area in foil to retain heat
Beginning the culture
5. In a sterile hood, pipette 200mL of TAP medium into a 500mL Erlenmeyer flask
6. Pipette 10mL of the Chlamydomonas strain into a 500mL Erlenmeyer flask containing the 200mL fresh medium (If the medium is grown in a slant tube, in a sterile field, use an inoculating loop to pull out some of the Chlamydomonas and place it in the 200 mL of fresh medium)
7. Parafilm seal the Erlenmeyer flask to prevent contamination and place the flask on a stir plate on top of the petri dish lid
8. Check it every so often to make sure there is continuous movement (YOU DO NOT WANT ANY SHADOW AREAS)
9. Set a timer to alternate between light and dark every 12 hours
10. Using a spectrophotometer measure the growth of the cell culture everyday at the same times (Set the spectrophotometer at 750nm and use identical Kimax tubes)
11. Incubate the cell culture until the growth curve reaches 0.5
12. Once the cell culture reaches 0.5 on the spectrophotometer and the culture’s cell density of approximately 0.9-2.0 x 107 ml-1 (3-5 days after transfer, late linear phase), aliquot the cells into 1.5mL Eppendorf tubes
Have liquid nitrogen ready in a foam container to snap freeze the aliquots
Culturing Chlamydomonas on solid medium
1. Set up a sterile field
2. Add 100uL of the liquid culture to TAP agar plates using a sterile rod/glass beads
3. Allow the plates to dry in the sterile field
4. Seal the plates in parafilm (this helps reduce the contamination)
5. Place the plates in the room temperature (25 degrees Celsius) incubator overnight
6. Remove the plates from the incubator and place them in a 4 degrees celsius refrigerator to prevent further growth
2. Add 100uL of the liquid culture to TAP agar plates using a sterile rod/glass beads
3. Allow the plates to dry in the sterile field
4. Seal the plates in parafilm (this helps reduce the contamination)
5. Place the plates in the room temperature (25 degrees Celsius) incubator overnight
6. Remove the plates from the incubator and place them in a 4 degrees celsius refrigerator to prevent further growth
Glass Bead Transformation of Chlamydomonas
Preparation
1. Grow cell culture to an OD750 of 0.15 to 0.4
2. Centrifuge at 400g for 5mins at room temperature 3. Resuspend in 1/100th the original volume of TAP 4. Add the following into a separate tube:
a. 8000PEG to 5% final concentration (10mg at 1mL of water is 1%)
b. 3ug of DNA
c. 0.3g of 500micron glass beads
d. 0.4mL Chlamy cell suspension
5. Mix with a pipette
6. Vortex at max speed for 15 seconds
Plating and Selection
1. Take 25uL of the cell suspension and add to 100uL of TAP with an appropriate antibiotic
2. Spread on a TAP plate with an appropriate antibiotic, using large glass beads
3. Allow the liquid to dry while avoiding light
4. Seal the plates with parafilm
5. Allow the colonies to grow (1-3 weeks)
6. Transfer the remaining cell/vortex culture to a 125mL flask with 20mL of TAP (w/o antibiotic)
7. Incubate for 6 hours on an orbital shaker at 70rpm
8. Add antibiotic to an appropriate concentration
9. Take 50uL of the cell suspension and spread on a TAP plate with an appropriate antibiotic with large glass beads
1. Grow cell culture to an OD750 of 0.15 to 0.4
2. Centrifuge at 400g for 5mins at room temperature 3. Resuspend in 1/100th the original volume of TAP 4. Add the following into a separate tube:
a. 8000PEG to 5% final concentration (10mg at 1mL of water is 1%)
b. 3ug of DNA
c. 0.3g of 500micron glass beads
d. 0.4mL Chlamy cell suspension
5. Mix with a pipette
6. Vortex at max speed for 15 seconds
Plating and Selection
1. Take 25uL of the cell suspension and add to 100uL of TAP with an appropriate antibiotic
2. Spread on a TAP plate with an appropriate antibiotic, using large glass beads
3. Allow the liquid to dry while avoiding light
4. Seal the plates with parafilm
5. Allow the colonies to grow (1-3 weeks)
6. Transfer the remaining cell/vortex culture to a 125mL flask with 20mL of TAP (w/o antibiotic)
7. Incubate for 6 hours on an orbital shaker at 70rpm
8. Add antibiotic to an appropriate concentration
9. Take 50uL of the cell suspension and spread on a TAP plate with an appropriate antibiotic with large glass beads
OD Spectrophotometer Readings (Prepare cuvettes in a Sterile Hood)
1. Set wavelength on a biochrom spectrophotometer to 750 nm
2. Blank spectrophotometer before each reading
a. Inside of a sterile hood pipette 3mL of TAP Media into 1 cuvette
b. Take cuvette with medium outside of hood
c. Wipe outside of cuvette thoroughly with KIMwipe
d. Insert into spectrophotometer and hit blank
3. OD Readings
a. Inside of a sterile hood pipette 2mL of TAP Media and 1mL of Chlamydomonas into one cuvette (3x)
b. Outside of the hood wipe the outside of the cuvette thoroughly with a KIMwipe and insert into spectrophotometer
c. Directly prior to reading, resuspend cells with pipette
d. Take reading
e. Multiply reading by three
f. Calculate the average
2. Blank spectrophotometer before each reading
a. Inside of a sterile hood pipette 3mL of TAP Media into 1 cuvette
b. Take cuvette with medium outside of hood
c. Wipe outside of cuvette thoroughly with KIMwipe
d. Insert into spectrophotometer and hit blank
3. OD Readings
a. Inside of a sterile hood pipette 2mL of TAP Media and 1mL of Chlamydomonas into one cuvette (3x)
b. Outside of the hood wipe the outside of the cuvette thoroughly with a KIMwipe and insert into spectrophotometer
c. Directly prior to reading, resuspend cells with pipette
d. Take reading
e. Multiply reading by three
f. Calculate the average
MEDIA RECIPES
SOC
*Make the media in a glass bottle with a screw off cap
1. Add 2g per 100mL of 2% Tryptone
2. Add 0.5g per 100mL of 0.5% Yeast Extract
3. Add 0.05g per 100mL of 10mM NaCl
4. Add 0.018g per 100mL of 2.5mM KCl
5. Add 80mL per 100mL of DI water
6. Adjust the pH to 7
7. Add 34mL of DI water
8. Autoclave for 15 minutes
9. Once cooled add 1mL per 100mL sterile MgCl2 in sterile hood
10. Add 2mL per 100mL of gluclose in sterile hood
1. Add 2g per 100mL of 2% Tryptone
2. Add 0.5g per 100mL of 0.5% Yeast Extract
3. Add 0.05g per 100mL of 10mM NaCl
4. Add 0.018g per 100mL of 2.5mM KCl
5. Add 80mL per 100mL of DI water
6. Adjust the pH to 7
7. Add 34mL of DI water
8. Autoclave for 15 minutes
9. Once cooled add 1mL per 100mL sterile MgCl2 in sterile hood
10. Add 2mL per 100mL of gluclose in sterile hood
LB Media
1. Mix 25g of LB Broth per 1000mL
2. Adjust pH to 7
3. Autoclave
2. Adjust pH to 7
3. Autoclave
LB Agar Plates
1. Add 15g agar to 1000mL LB Media
2. Add 1uL of antibiotic per 1mL
3. When cool to touch, poor 20mL into each plate in a sterile field
2. Add 1uL of antibiotic per 1mL
3. When cool to touch, poor 20mL into each plate in a sterile field
TAP Agar Plates
1. Pour 500mL of TAP into a 1L Erlenmeyer flask
2. Add 7.5g of agar
3. Stir for 10mins
4. Autoclave
5. Add appropriate antibiotic
2. Add 7.5g of agar
3. Stir for 10mins
4. Autoclave
5. Add appropriate antibiotic
Buffers
Miniprep Buffers
Re-suspension buffer (equivelent of Qiagen Buffer P1)
- Tris – HCl-50mM
- EDTA – 10mM
- RNase A- 100g/ml
- HCl – final pH8 (Note: store RNase A @ -20 degrees C, aliquot buffer and add at time of use, do not autoclave)
Lysis buffer
- NaOH – 200mM
- SDS – 1% (w/v) (Note: do not autoclave SDS, use sterile filter)
Neutralization Buffer (equivalent of Qiagen Buffer PB)
- GU-HCl – 4.2 M
- KOAc – 0.9 M
- HOAc – final pH 4.2
Column Wash/Binding Buffer (equivalent of Qiagen Buffer PB)
- GU – 5.0 Ml
- sopropanol – 30% (v/v)
Column Wash Buffer (equivalent of Qiagen Buffer PE)
- Tris-HCl – 10mM
- Ethanol – 80% (v/v)
- HCl – final pH 7.5
Mini-column Recycling buffer(Equilibrium buffer (equivalent of Qiagen Buffer QBT))
- NaCl – 750mM
- MOPS – 50mM Isopropanol – 15% (v/v)
- Triton X-100 – 0.15% (v/v) (Note: do not autoclave MOPS)
Protein Purification Buffer (NID Extraction Buffer)
- EDTA – 20-50 mM
- Tris – HCl – 50mM
- NH4Cl -0.75 M
- Triton X-100 – 0.5% (v/v)
- Sucrose – 5% (w/v)
- Lysozyme - 100g/ml
- RNase A - 25g/ml
- HCL – final pH 8
(Note: Store RNase A and Lysozyme @ -20 degrees Celsius. Aliquot buffer and add at time of use, do not autoclave)
Re-suspension buffer (equivelent of Qiagen Buffer P1)
- Tris – HCl-50mM
- EDTA – 10mM
- RNase A- 100g/ml
- HCl – final pH8 (Note: store RNase A @ -20 degrees C, aliquot buffer and add at time of use, do not autoclave)
Lysis buffer
- NaOH – 200mM
- SDS – 1% (w/v) (Note: do not autoclave SDS, use sterile filter)
Neutralization Buffer (equivalent of Qiagen Buffer PB)
- GU-HCl – 4.2 M
- KOAc – 0.9 M
- HOAc – final pH 4.2
Column Wash/Binding Buffer (equivalent of Qiagen Buffer PB)
- GU – 5.0 Ml
- sopropanol – 30% (v/v)
Column Wash Buffer (equivalent of Qiagen Buffer PE)
- Tris-HCl – 10mM
- Ethanol – 80% (v/v)
- HCl – final pH 7.5
Mini-column Recycling buffer(Equilibrium buffer (equivalent of Qiagen Buffer QBT))
- NaCl – 750mM
- MOPS – 50mM Isopropanol – 15% (v/v)
- Triton X-100 – 0.15% (v/v) (Note: do not autoclave MOPS)
Protein Purification Buffer (NID Extraction Buffer)
- EDTA – 20-50 mM
- Tris – HCl – 50mM
- NH4Cl -0.75 M
- Triton X-100 – 0.5% (v/v)
- Sucrose – 5% (w/v)
- Lysozyme - 100g/ml
- RNase A - 25g/ml
- HCL – final pH 8
(Note: Store RNase A and Lysozyme @ -20 degrees Celsius. Aliquot buffer and add at time of use, do not autoclave)
