Subcloning

From Genome@Yale

Notes and protocols by Laura DeMare & Tae Hoon Kim

Contents 1 Primer Design 2 PCR 3 TOPO cloning 4 Restriction Digest 5 Ligation 6 Verification of Clones 7 Transformation 8 Miniprep 9 Competent Cells 10 Plates & Media 10.1 2xYT 10.2 LB agar plates 10.3 GAL plates 10.4 DOG plates

Primer Design

PCR

TOPO cloning

Restriction Digest

General considerations:

• double digest chart
• double digest calculator
• enzyme lookup
• buffer chart
• Look up restriction endonuclease in NEB catalog
  • Buffer and % activity; use buffer with highest activity
• For double digests, check which buffer is recommended
  • Incubation temperature (usually 37°C)
  • Activity (in units, where 1 unit = digestion of 1 ug DNA in 1 hour in 50 ul)
  • 2,000 units: ~overnight (for complete digestion)
  • 10,000 units: ~ 2 hours

Other notes:

• Isoschinzomers: enzymes that recognize the same sequence
• Cleavage @ ends of linearized fragments may be less efficient
• DNA methylation can block digestion (enzyme-specific)

For digestion of backbone (for cloning) ~ 5 ug DNA total:

20 ul rxn volume
5 ul DNA (for 1 mg/ml stock)
2 ul 10X buffer (should not be >10% rxn volume)
1.8 ul enzyme (or 0.9 ul/enzyme if double digest)
rest ddH2O 

1. Flick to mix, incubate at 37°C
2. 20-60 minutes for analytic/incomplete digestion
3. Up to overnight for complete digestion
4. Purify digested DNA (Qiagen PCR purification and elute in 30 ul OR heat inactivate enzyme)

For sequential digest (i.e. buffers not compatible), eluted DNA is subjected to second digest:

40 ul rxn volume
30 ul eluted DNA
4 ul buffer
3.6 ul enzyme
rest ddH2O (5.4 ul)

1. Flick to mix, incubate at 37°C
2. Purify digestion

Notes:

• If linearized DNA is going to be used for cloning, must gel purify (insert and/or vector, depending on size desired) for correct fragment
• To prevent recircularization, can treat with phosphatase (be careful, could affect ligation if not properly inactivated/purifed)

Ligation

10 ul rxn volume
1 ul (or 2) vector DNA (~50 ng)
7 ul insert DNA
1 ul 10X ligase buffer (*if flakes, shake/heat to dissolve precipitate)
0.9 ul T4 DNA ligase

1. Flick to mix, incubate at 16°C overnight (shake for first 20 min)
2. After 1 hour, test ligation by removing 3 ul for transformation
3. Transform into bacteria, grow on antibiotic plates for selection
4. Negative control: cut vector, no insert (linear DNA→ few colonies)
5. Positive control: uncut vector (circular DNA → many colonies)
6. Count colony growth; compare to negative control

Verification of Clones

• Ligate, transform, grow and count colonies

1. Pick twice as many colonies as statistically necessary to select a positive clone
2. If less colonies than negative control, pick all
3. Max number that can be mini-prep'd: ~20
4. Grow starter colonies in 2 ml media (+selection) in 14 ml round-bottom tube
5. Use 1 ml for purification, save rest in 4°C
6. Purify plasmids from each colony (Qiagen Mini-prep)
7. Double digest (with sites on either side of insert) in 10 ul rxn volume (8 ul DNA, 1 ul buffer, 0.5 ul/enzyme, for 1 hour)
8. To check fragment size for insertion, run on gel
9. For properly cloned colonies, use 0.5 ml of saved starter colony to grow up in flask for midi-prep and other 0.5 for freezer stock (add 80% glycerol)

Transformation

For competent DH5α E. coli (store in -80°C) and plasmid (circular) DNA

• plasmid DNA: 50 ng/ml (1:20 dilution of 1 mg/ml stock)
• competent cells: defrost in ice bucket immediately before use

1. Add 1 ul plasmid and 50 ul cells in eppendorf, flick to mix
2. Incubate on ice for 30 minutes
3. Defrost LB bacterial plate (with desired antibiotic) in 37°C
4. Last 5 minutes- add H2O to well in 42°C heat block
5. Heat shock tube for 30 seconds at 42°C
6. Put back on ice for ~2 minutes
7. Plate cells (all 50 ul), add 5-6 beads and shake
8. Grow colonies for ~16 hours in 37°C incubator

Notes:

• Adjust the concentration of DNA to increase/decrease transformation rate
• To prevent growth, put plates in 4°C

Miniprep

Miniprep or starter culture for maxipreps

• LB media + 100 ug/ml Ampicillin (or other antibiotic)

1. Add 2mL media to 14 ml Falcon round-bottom tube
2. Pick single colony with micropipet tip and transfer to tube by pipetting up and down
3. Put lid loosely on tube
4. Incubate and shake at 37°C for 6-18 hours (media should turn cloudy)
5. Transfer 1 ml to larger flask with media (50-100 ml)
6. Cover with aluminum foil
7. Incubate and shake at 37°C overnight (up to ~20 hours)

• Harvest bacteria by centrifuging @ 4000 rpm for 15 minute at 4°C
• Plasmid purification (Qiagen- mini/midi/maxi)
• helpful hints for midipreps

Competent Cells

Nishimura, A.,Morita, A., Nishimura, Y., and Sugino, Y. A rapid and highly efficient method for preparation of competent Escherichia coli cells. (1990) Nucleic Acid Res 18:20, p 6169.

1. Inoculate 100ml of medium A with single colony or 0.5ml culture.
2. Grow to mid log phase @37°C (OD(600)=0.4-0.8)
3. Keep on ice for 10 minutes
4. Pellet cells at 1500 x g for 10 minutes at 4°C.
5. Resuspend pellet in 1.0 ml of medium A precooled on ice.
6. Then add 5.0 ml of storage solution B.
7. Divide into aliquots of 0.10 ml each and store in -80°C (quick freeze is optional)

Medium A: LB supplemented with 10 mM MgSO₄-7H₂O and 0.2% Glucose

stock         for 50mL         for 100mL
2XLB          25mL             50mL
1M MgSO4      0.5mL            1mL
45% glucose   0.22mL           0.44mL
dH2O          24.28mL          48.56mL

Medium B: 25% glycerol, 12% PEG (MW7500), 12 mM MgSO₄-7H₂O added to LB and sterilized through filtration

stock         for 50mL
2XLB          25mL
1M MgSO4      0.5mL
50% PEG       12mL
100% glycerol 12.5mL

Plates & Media

2xYT

components       for 1L     for 4L
Bactotryptone    16g        64g
Yeast Extract    10g        40g
NaCl             5g         20g

1. Dissolve completely
2. Autoclave in 1L bottles

LB agar plates

components       for 1L
Bactotryptone    10g
Yeast Extract    5g
NaCl             10g
Bactoagar        15g

1. Autoclave, let it cool to 50°C (touchable to inner forearm)
2. Add antibiotics (for AMP100, add 2mL 50mg/mL stock; for KAN50, add 5mL 10mg/mL stock)
3. Pipet 20mL per each plate and use flame to remove bubbles
4. Let plates sit O/N to dry and cool completely

GAL plates

1. Autoclave 7.5g agar in 400mL dH2O
2. Add 100mL 5X M63 medium
3. Add 0.5mL 1M MgSO4
4. Let it cool to 50°C (touchable to inner forearm) and add the following

Stock            for 0.5L
20% galactose    5mL
0.2mg/mL biotin  2.5mL
10mg/mL leucine  2.25mL

1. Add antibiotics (for AMP100, add 1mL 50mg/mL stock; for CM12.5, add 184uL 34mg/mL stock)
2. Pipet 20mL per each plate and use flame to remove bubbles
3. Let plates sit O/N to dry and cool completely

DOG plates

1. Autoclave 7.5g agar in 400mL dH2O
2. Add 100mL 5X M63 medium
3. Add 0.5mL 1M MgSO4
4. Let it cool to 50°C (touchable to inner forearm) and add the following

Stock               for 0.5L
20% deoxygalactose  5mL
20% glycerol        5mL
0.2mg/mL biotin     2.5mL
10mg/mL leucine     2.25mL

1. Add antibiotics (for AMP100, add 1mL 50mg/mL stock; for CM12.5, add 184uL 34mg/mL stock)
2. Pipet 20mL per each plate and use flame to remove bubbles
3. Let plates sit O/N to dry and cool completely