Editing Lambda Red Protocol (section)

== Procedure ==

*'''General outline'''
**Grow up pKD46, pKD3, and pCP20 in host strains
***pKD46 should be grown at <= 32oC to maintain the temperature sensitive plasmid.
**Perform minipreps to extract plasmids
**Transform pKD46 into target strain, plate out on LB-amp plates
**PCR amplify linear fragment from pKD3 or pKD4 using oligos ''A'' and ''B'' (see below for design)
**Make target strain (now maintaining pKD46) electrocompetent by growing at 30°C with L-arabinose
**Electroprate linear DNA into electrocompetent cells
**Grow at 37°C on chloramphenicol plates
**PCR verify the deletion with oligos ''C'' and ''D'' (see below for design)
*'''Detailed procedure'''
*'''Day 0: Start overnight culture'''
**Start overnight culture of strain containing gene to knock out.
*'''Day 1: Preparation and transformation of competent cells'''
**Make new glycerol stock of overnight strain (grown from single colony)
**Add 300 {{ul}} overnight culture to 30 mL LB medium (1:100 dilution)
**Check culture density every 30 minutes starting at +1 hour; grow to {{od|600}} of 0.3 to 0.4
**{{od|600}} measurements of K91
***+2:00 hrs: 0.06
***+2:45 hrs: 0.3008
**Spin at 2500rcf for 10 minutes at 4°C in two 50 mL centrifuge tubes (JA-20 rotor)
**Decant supernatant, discard
**Resuspend each pellet in 5 mL ice cold transformation buffer; swirl or pipette gently to mix
***'''Transformation Buffer'''
***10 mM Pipes
***15 mM {{cacl2}}
***250 mM KCl
***Titrate to pH 6.7 before adding {{mncl2}}
***55 mM {{mncl2}}
***Filter sterilize
**Incubate on ice for 10 min
**Spin at 2500rcf for 10 minutes at 4°C
**Decant supernatant, discard
**Resuspend each pellet in 1.25 mL ice cold transformation buffer
**Combine resuspended pellets in single tube
**Remove 400 {{ul}} for immediate transformation
**Add DMSO to a final concentration of 7% (160 {{ul}}). Drip the DMSO slowly into the cell suspension, with constant swirling by hand.
**Incubate on ice for 10 min
**Aliquot 400 {{ul}} each into five 1.5 mL tubes
**Store in -80°C freezer.
**Transform strain with pKD46 and grow on LB-amp plate at 30°C
***Prepare four tubes with 0, 1 ng, 10 ng, and 100 ng pKD46 plasmid DNA
***Add 100 {{ul}} of competent cell mix to each tube
***Incubate on ice 30 min
***Heat shock 30 seconds at 42°C
***Incubate on ice 2 min
***Spread all 100 {{ul}} on LB-amp-kan plate
***Incubate at 30°C overnight
**U45endA—pKD3[Cat]—D45endA fragment is chloramphenicol cassette with FRT sequences, flanked by 45 bp upstream and downstream of endA.
**PCR U45endA— pKD3[Cat]—D45endA from pKD3 and verify on gel
***PCR program: 95°C 7min → 35*[94°C 15s → 50°C 30s → 72°C 90s]

::

{|
|-
| align="center" style="background:#f0f0f0;" | '''Contents'''
| align="center" style="background:#f0f0f0;" | '''Concentration'''
| align="center" style="background:#f0f0f0;" | '''Volume'''
|-
| pKD3 template
| 45 ng/{{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
| 0.5 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| forward primer
| 10 {{um}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">&nbsp;μM</span>
| 5 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| reverse primer
| 10 {{um}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">&nbsp;μM</span>
| 5 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| 10x KOD buffer
| -
| 10 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| dNTP
| 2 mM
| 10 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| {{mgso4}}
| 25 mM
| 4 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| KOD polymerase
| 
| 2 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|-
| d{{h2o}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">H</span><sub style="line-height: 1; color: rgb(0, 0, 0); font-family: sans-serif;">2</sub><span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">O</span>
| -
| 64.5 {{ul}}<span style="color: rgb(0, 0, 0); font-family: sans-serif; font-size: 12.7px;">μL</span>
|}



*
*Make ten 10 {{ug}}/ml chloramphenicol plates and ten 25 {{ug}}/ml chloramphenicol plates

*'''Day 2'''
**Make 1 M stock of L-arabinose
***MW of L-arabinose is 150.13
***Add 1501.3 mg of L-arabinose to 8.5 g d{{h2o}} to make 1 M stock
**Retrieve plates from incubator
**Check results from the transformation
***1 ng pKD46 transformation yielded about 10 colonies
***10 ng pKD46 transformation yielded about 100 colonies
***100 ng pKD46 transformation yielded several hundred colonies
**Pick some colonies and grow at 30°C in 2 mL LB + 50 {{ug}}/mL Amp
***add 50 {{ul}} 10 mg/mL Ampicillin stock
**Include enough samples for two conditions: +/- L-arabinose induction
**When {{od|600}} of cells(+pKD46) reaches 0.1, add L-arabinose to concentration of 10 mM to induce pKD46 λ-red expression
***add 20 {{ul}} 1 M L-arabinose to 2 mL culture
**Continue to grow at 30°C to {{od|600}} = 0.4
**Aliquot 1 mL each into two 1.5 mL centrifuge tubes
**Chill cells in ice-water bath 10 minutes
**Centrifuge 10 min at 4000rcf 4°C
**Pipette off supernatant and resuspend pellets in 1 mL ice-cold d{{h2o}}
**Centrifuge 10 min at 4000rcf 4°C
**Resuspend pellet in 50 {{ul}} d{{h2o}}
**For electroporation step, include 2 conditions: +/- PCR fragment
**Chill electroporation cuvettes for 5 minutes on ice
**Add 5 pg to 0.5 {{ug}} PCR amplified DNA to cells
**Set electroporation apparatus to 2.5 kV, 25 μF. Set the pulse controller to 200 ohms
**Place the cuvette into the sample chamber
**Apply the pulse by pushing the button
**Remove the cuvette. Immediately add 1 mL LB medium and transfer to a sterile culture tube
**Incubate 60-120 min with moderate shaking at 37°C
**Plate aliquots of the transformation culture on LB plates supplemented with chloramphenicol (10 {{ug}}/mL, 25 {{ug}}/mL)