Haynes:Assembly101: Difference between revisions
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<- [[Haynes:Protocols | Back to Protocols]] | |||
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<font size=3>'''Model Procedure for Assembling Parts: Classic Ligation for Beginners'''</font><br> | <font size=3>'''Model Procedure for Assembling Parts: Classic Ligation for Beginners'''</font><br> | ||
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Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.<br> | Day 1*: Pick and amplify the desired plasmid DNA by growing transformed [http://openwetware.org/wiki/E._coli_genotypes#DH5.CE.B1_Turbo_.28NEB.29 DH5α Turbo] bacteria.<br> | ||
'''Make streaks from glycerol stocks''' ''6 hours'' | '''Make streaks from glycerol stocks''' ''6 hours'' | ||
# Warm an agar plate at 37°C for at least 20 min. | # Warm an agar plate at 37°C for at least 20 min. | ||
# Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date. | # Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date. | ||
# Locate the desired -80°C glycerol stock. Use a sterile wooden toothpick to scrape up a tiny bit of the frozen bacteria and streak the plate. | # Locate the desired -80°C glycerol stock. Use a sterile wooden toothpick or plastic micropipette tip to scrape up a tiny bit of the frozen bacteria and streak the plate. | ||
# Incubate the plate at 37°C for 6 hours to grow the bacteria.<br<br> | # Incubate the plate at 37°C for 6 hours to grow the bacteria.<br><br> | ||
'''Grow liquid cultures''' | '''Grow liquid cultures''' | ||
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To extract the plasmid DNA from the bacteria, perform a mini prep (refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl). | To extract the plasmid DNA from the bacteria, perform a mini prep (refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl). | ||
{| class="wikitable" width=400px | {| class="wikitable" width=400px align="right" | ||
| Plasmid DNA || 15.0 μl* | | Plasmid DNA || 15.0 μl* | ||
|- | |- | ||
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| colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 37°C for 10 minutes. | | colspan="2" | *For low yield DNA, use up to 25 μL; decrease dH<sub>2</sub>O accordingly.<br>Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at 37°C for 10 minutes. | ||
|} | |} | ||
<br> | '''Digest (cut) the DNA with restriction enzymes''' ''30 minutes'' | ||
# First, write out a brief assembly strategy: <br>''New Construct Name'': ''BioBrick Insert Name'', size (bp), cut sites + ''BioBrick Vector Name'', size+backbone (bp), cut sites | |||
# Set up your digest reaction(s) as shown to the right: | |||
<br><br><br><br><br><br><br> | |||
'''Separate the fragments via gel electrophoresis and purify the fragments''' ''2 hours'' | '''Separate the fragments via gel electrophoresis and purify the fragments''' ''2 hours'' | ||
[[Image:KAH_011711_gel2.tif|thumb|350px|Blue dashed lines border where the gel was cut to excise vector fragments (lanes 1 and 2) and insert fragments (lanes 3 and 4).]] | |||
# Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask. | # Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask. | ||
# Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds. | # Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds. | ||
# Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample. | # Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample. | ||
# Add | # Add 6 μl of SYBR safe stain. Mix by swirling (avoid making bubbles). | ||
# Pour the gel into the gel mold. Allow it to cool until it becomes opaque. | # Pour the gel into the gel mold. Allow it to cool until it becomes opaque. | ||
# Fill a gel electrophoresis chamber with 1x TAE. | # Fill a gel electrophoresis chamber with 1x TAE. | ||
# | # Gently remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber. | ||
# Carefully pipette | # Carefully pipette 3-6 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells. | ||
# Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V. | # Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V. | ||
# Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.). | # Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.). | ||
# Remove the gel from the chamber and photograph under UV light. | # Remove the gel from the chamber and photograph under UV light. | ||
# Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA (refer to the Qiagen gel purification protocol; elute with 30 μL EB buffer). | # Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA (refer to the Qiagen gel purification protocol; elute with 30 μL EB buffer). | ||
# Measure the concentration of the purified fragment samples with a Nanodrop Spectrophotometer. | # Measure the concentration of the purified fragment samples with a Nanodrop Spectrophotometer. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample. | ||
<br> | <br> | ||
{| class="wikitable" width=400px align="right" | |||
{| class="wikitable" width= | |||
| || Ligation || Negative Control | | || Ligation || Negative Control | ||
|- | |- | ||
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| 2x Roche Rapid Ligation buffer || 5.0 μl || same | | 2x Roche Rapid Ligation buffer || 5.0 μl || same | ||
|- | |- | ||
| | | New England Biolabs T4 ligase || 1.0 μl || same | ||
|- | |- | ||
| dH<sub>2</sub>O || ___ μL || ___ μL + | | dH<sub>2</sub>O || ___ μL || ___ μL + ''Insert'' μL | ||
|- | |- | ||
| || 10.0 μL total || same | | || 10.0 μL total || same | ||
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| colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | | colspan="3" | Mix the reaction(s) thoroughly by flicking the tube.<br>Incubate at room temperature for 10 minutes. | ||
|} | |} | ||
'''Ligate (paste) the DNA fragments together''' ''15 minutes'' | |||
# Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules<br>''X ng insert = (bp insert / bp vector) x 2 x 50 ng vector'' | |||
# Calculate how many μL of insert and vector you will need for each ligation:<br>''X μL insert = desired ng insert ÷ insert concentration ng/μL'' <br>''X μL vector = 50 ng vector ÷ vector concentration ng/μL'' | |||
# Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown here: | |||
<br><br> | <br><br> | ||
'''Transform bacteria with the ligated plasmids''' '' | '''Transform bacteria with the ligated plasmids''' ''60 minutes'' | ||
# Warm selection agar plates at 37°C. | |||
# Incubate | We'll be following the traditional transformation protocol described [http://openwetware.org/wiki/Haynes:TransformationPlasmids#Traditional_Method:_Chemically_competent_cells_.2B_ligation here]. | ||
# Add 30 μL thawed cells to the ligation reaction. | # Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min. | ||
# | # Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation. | ||
# Pipette the total volume of cells | # Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation). | ||
# Incubate overnight at 37°C to get colonies | # Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down '''gently''' to mix the cells and DNA. | ||
# Incubate on ice for 10 min. | |||
# Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute. | |||
# Add 750 μL sterile SOC medium to each sample. | |||
# Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes. | |||
# Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period. | |||
# Pellet the cells by centrifugation at top speed for 3 minutes. | |||
# Discard the SOC supernatant. | |||
# Resuspend the pellet in 100 μL LB medium (plus proper antibiotic). | |||
# Pipette the total volume of cells onto the agar; spread using sterile glass beads. | |||
# Incubate the inverted plate(s) overnight at 37°C to get colonies. | |||
Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted). | |||
Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. | Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure. | ||
</div> | </div> | ||
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'''Check the plates, grow cultures, and do minipreps''' ''6 hours'' | '''Check the plates, grow cultures, and do minipreps''' ''6 hours'' | ||
# Compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. | # Compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies. | ||
# If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures (see Day 1, '''Grow liquid cultures'''). If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation. | # If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures (see Day 1, '''Grow liquid cultures'''). Grow for 5 - 6 hours.<br>''If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation.'' | ||
# Miniprep the DNA from the liquid cultures (see Day | # Miniprep the DNA from the liquid cultures (see Day 2, '''Extract the plasmid DNA: Qiagen Miniprep Kit''') | ||
# Digest 2 uL of each DNA sample with EcoRI/ PstI and check via gel electrophoresis (1% agarose) to confirm the assembled construct size. You should see one fragment that is the backbone, and another fragment that equals the total size of the two BioBrick parts you assembled. | # Digest 2 uL of each DNA sample with EcoRI/ PstI and check via gel electrophoresis (1% agarose) to confirm the assembled construct size. You should see one fragment that is the backbone, and another fragment that equals the total size of the two BioBrick parts you assembled. | ||
</div> | </div> |
Latest revision as of 10:57, 18 November 2015
Model Procedure for Assembling Parts: Classic Ligation for Beginners
or, Cloning Sensei's Guide For the Aspiring Cloning Ninja
Day 1*: Pick and amplify the desired plasmid DNA by growing transformed DH5α Turbo bacteria.
Make streaks from glycerol stocks 6 hours
- Warm an agar plate at 37°C for at least 20 min.
- Label the plate with the bacterial strain name (e.g., DH5α), the antibiotic, the BioBrick part(s) name, your initials, and the date.
- Locate the desired -80°C glycerol stock. Use a sterile wooden toothpick or plastic micropipette tip to scrape up a tiny bit of the frozen bacteria and streak the plate.
- Incubate the plate at 37°C for 6 hours to grow the bacteria.
Grow liquid cultures
- Label 15 ml sterile culture tube(s) appropriately. Fill each tube with 2 ml of LB growth medium + appropriate antibiotic (e.g., 100 μg/ml ampicillin).
- Using a sterile pipette tip, touch the bacterial streak (or pick up a single colony) and put the tip into the LB medium (bacterial end down).
- Grow the cultures overnight in a shaking 37°C incubator.
Day 2: Extract the plasmids. Digest (cut), purify, and ligate (paste) the BioBricks. Put the assembled plasmid into bacteria
Extract the plasmid DNA: Qiagen Miniprep Kit 1.5 hours
To extract the plasmid DNA from the bacteria, perform a mini prep (refer to the Qiagen miniprep protocol). 2 ml of culture usually gives a yield of about 200 ng/μl (elution vol. = 75 μl).
Plasmid DNA | 15.0 μl* |
Fermentas FastDigest enzyme 1 | 1.0 μl |
Fermentas FastDigest enzyme 2 | 1.0 μl |
10x FastDigest buffer + green loading dye | 3.0 μl |
dH2O | 10.0 μl |
30.0 μl total | |
*For low yield DNA, use up to 25 μL; decrease dH2O accordingly. Mix the reaction(s) thoroughly by flicking the tube. Incubate at 37°C for 10 minutes. |
Digest (cut) the DNA with restriction enzymes 30 minutes
- First, write out a brief assembly strategy:
New Construct Name: BioBrick Insert Name, size (bp), cut sites + BioBrick Vector Name, size+backbone (bp), cut sites - Set up your digest reaction(s) as shown to the right:
Separate the fragments via gel electrophoresis and purify the fragments 2 hours
- Make a 0.8% gel: add 0.48 g agarose to ~60 ml 1x TAE buffer in a glass flask.
- Mix by swirling and microwave for 40 seconds. Mix by swirling again (to eliminate air pockets and prevent boiling-over) and microwave for 40 seconds.
- Set up a gel mold and comb. Make sure the teeth are the right size to hold 30 μL of sample.
- Add 6 μl of SYBR safe stain. Mix by swirling (avoid making bubbles).
- Pour the gel into the gel mold. Allow it to cool until it becomes opaque.
- Fill a gel electrophoresis chamber with 1x TAE.
- Gently remove the comb from the gel and carefully submerge the gel into the filled electrophoresis chamber.
- Carefully pipette 3-6 μL pre-made 1 kb ladder mix into the first empty well and the DNA samples into the other empty wells.
- Connect the electrical leads so that the positive end is at the bottom (DNA migrates to the positive end). Run the gel at 100 V.
- Stop the gel when the yellow dye (Orange G) reaches the desired place on the gel (~1 hr.).
- Remove the gel from the chamber and photograph under UV light.
- Use a scalpel to cut the appropriate sized band(s) from the gel, place each gel slice in a 1.5 mL tube, and purify the DNA (refer to the Qiagen gel purification protocol; elute with 30 μL EB buffer).
- Measure the concentration of the purified fragment samples with a Nanodrop Spectrophotometer. Record the absorbance (A260), purity (A260/A280), and concentration (ng/μl) for each sample.
Ligation | Negative Control | |
Insert DNA (X ng) | ___ μL | none |
Vector DNA (50 ng) | ___ μL | same |
2x Roche Rapid Ligation buffer | 5.0 μl | same |
New England Biolabs T4 ligase | 1.0 μl | same |
dH2O | ___ μL | ___ μL + Insert μL |
10.0 μL total | same | |
Mix the reaction(s) thoroughly by flicking the tube. Incubate at room temperature for 10 minutes. |
Ligate (paste) the DNA fragments together 15 minutes
- Calculate how many ng of insert you need to get a 2:1 ratio of insert molecules to 50 ng vector molecules
X ng insert = (bp insert / bp vector) x 2 x 50 ng vector - Calculate how many μL of insert and vector you will need for each ligation:
X μL insert = desired ng insert ÷ insert concentration ng/μL
X μL vector = 50 ng vector ÷ vector concentration ng/μL - Set up your ligation reaction(s) in sterile 0.5 mL tubes as shown here:
Transform bacteria with the ligated plasmids 60 minutes
We'll be following the traditional transformation protocol described here.
- Warm selection agar plates at 37°C (one for each plasmid, plus one for a zero plasmid control) for at least 15 min.
- Incubate chemically competent cells on ice just until thawed. You will need 30 μL cells per ligation.
- Add 30 μL thawed cells to to 1 sterile 2.0 mL tube (per ligation).
- Add the total ligation reaction to the cells in each 2.0 mL tube. Pipette up and down gently to mix the cells and DNA.
- Incubate on ice for 10 min.
- Heat shock: Transfer the tubes to 42°C for exactly 45 seconds (heat shock) on a heat block or water bath, then immediately place the tubes on ice for 1 minute.
- Add 750 μL sterile SOC medium to each sample.
- Recovery: Close the caps tightly. Place the tubes in the shaking incubator, secured in a sideways position with tape. Incubate the tubes, with shaking, at 37°C for 45 minutes.
- Pre-warm the agar plates: Incuabte the selection agar plates (one per sample) at 37°C during the 45 minute recovery period.
- Pellet the cells by centrifugation at top speed for 3 minutes.
- Discard the SOC supernatant.
- Resuspend the pellet in 100 μL LB medium (plus proper antibiotic).
- Pipette the total volume of cells onto the agar; spread using sterile glass beads.
- Incubate the inverted plate(s) overnight at 37°C to get colonies.
Note: To store the colonies long term, seal the plate with parafilm and keep the plate at 4°C (inverted). Note: The negative control will show you the number of “background” colonies so that you can determine whether your transformation worked, or is just the result of vector self-ligation or selection failure.
Day 3: Confirm the assembly
Check the plates, grow cultures, and do minipreps 6 hours
- Compare the plates to estimate the ratio of “ligation” colonies to “negative control” colonies.
- If the ratio is 10:1 or greater, great job! Pick 2 colonies for separate liquid cultures (see Day 1, Grow liquid cultures). Grow for 5 - 6 hours.
If the ratio is less than 10:1, pick more colonies or trouble shoot and repeat the ligation & transformation. - Miniprep the DNA from the liquid cultures (see Day 2, Extract the plasmid DNA: Qiagen Miniprep Kit)
- Digest 2 uL of each DNA sample with EcoRI/ PstI and check via gel electrophoresis (1% agarose) to confirm the assembled construct size. You should see one fragment that is the backbone, and another fragment that equals the total size of the two BioBrick parts you assembled.
Timeline
- Level 1, Newbie: Undergraduates and unseasoned scientists can expect to spend a week to two weeks on one assembly step. You will inevitably spill something, forget a step, plan an assembly incorrectly, or mess up some other inventive way. Or you have classes and can't spend every day in the lab.
- Level 2, Graduate Student: Typically have experience pipetting and handling samples well and can expect to spend 3 days per assembly. If you have no life and are super-ambitious, you can crank out an assembly cycle in two days (when Day 2 procedures are started immediately after the Day 3 procedures in a single day), and complete three assemblies in one week.
- Level 3, Postdoc "Cloning Ninja": If you have no life, are super-impatient, and are trying to publish papers, you can crank out an assembly cycle in two days, and complete three assemblies in one week.