Knockout1

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	| align="center" style="background:#f0f0f0;"|'''Volume'''		| align="center" style="background:#f0f0f0;"|'''Volume'''
	|-		|-
-	| pKD3 template||45 ng/{{ul}}||0.5 {{ul}}	+	| pKD13 template||45 ng/㎕||1㎕
	|-		|-
-	| forward primer||10 {{um}}||5 {{ul}}	+	| forward primer||10 pmol||2㎕
	|-		|-
-	| reverse primer||10 {{um}}||5 {{ul}}	+	| reverse primer||10 pmol||2㎕
	|-		|-
-	| 10x KOD buffer||-||10 {{ul}}	+	| 10x EX buffer||-||5㎕
	|-		|-
-	| dNTP||2 mM||10 {{ul}}	+	| dNTP||2 mM||2㎕
	|-		|-
-	| {{mgso4}}||25 mM||4 {{ul}}	+	| EX-Tag polymerase||-||0.5㎕
	|-		|-
-	| KOD polymerase||||2 {{ul}}	+	| d{{H2O}}||-||50㎕
-	|-	+
-	| d{{h2o}}||-||64.5 {{ul}}	+
	|}		|}

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Revision as of 09:05, 21 October 2010

Contents 1 Gene replacement 1.1 Materials 1.2 Procedure 1.3 Notes

Gene replacement

• target gene: yfaL

Materials

• Plasmids
  • pKD46, pKD13, pCP20 - see this link

• Target gene
  • yfaL
    • an AmpR and CmR plasmid that shows temperature-sensitive replication and thermal induction of FLP synthesis (Datsenko and Wanner paper [1])

• Reagents
  • L-arabinose
    • Lambda red recombinase pBAD promoter inducer
• Equipment
  • Incubators (30°C and 37°C)
  • Electroporator

Procedure

• Preparation of plasmids

1. Grow up pKD46, pKD13, and pCP20 in proper host strains
2. Perform minipreps to extract plasmids

• Transformation of pKD46 to target strain
  • Preparation of competent cells
    • Using the protocol of TSS method - TSS method.
    • Target strain - BW25113, Top10

• Generation of cassette flanked by homologous arms
  • Design of primers
    • Primers have overhangs which were homologous to surrounding region of target gene.
    • Sequences from cassette region have about 60℃ Tm-value.
  • PCR condition
    • Anneling temparature and extension time depends on the target sequence.
    • Anneling temparature is tested at 55℃ at first.
    • Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
  • PCR program
    • 94°C 1min → 30*[98°C 11s → 55°C 30s → 72°C 2min] → 72°C 5min

Contents	Concentration	Volume
pKD13 template	45 ng/㎕	1㎕
forward primer	10 pmol	2㎕
reverse primer	10 pmol	2㎕
10x EX buffer	-	5㎕
dNTP	2 mM	2㎕
EX-Tag polymerase	-	0.5㎕
dTemplate:H2O	-	50㎕

• Making competent cells

1. Pick the single colony containg pKD46.
2. Inoculate it to 5㎖ LB broth with ampicilline and L-arabinose.
   • L-arabinose final concentration is 1mM.
3. Incubate at 27℃, 180rpm until the culture OD reaches 0.6.
4. Centrifuge 3600rpm, for 5min at 4℃.
5. Wash with ice-cold 10% glycerol 3 times.
6. Concetrate 100-fold.

• Electroporation

1. Use 30㎕ competent cell and 300ng PCR product.
2. Pulse 2.5kV, 200Ω, 25㎌.
3. Add prewarmed 1㎖ LB broth immediately.
4. Incubate 2 hour at 37℃.
5. Spread one-tenth of the sample and incubate at 37℃.

Notes

• Primers
  • For pKD 13 kanamycin cassette
    • Foward: ATGCGGATTATCTTTCTACGCAAGGAGTATTTATCTTTACaATTCCGGGGATCCGTCGACC
    • Reverse: gcgcccctgccagcacgtagtgtgtaggctggagctgcttcggccggttagcgccatcggct
  • For confirmation of Knockout
    • Foward: GTTTCATGCAGACTACCTGTAGTACG
    • Reverse: ctgcctcgtcctgcagttcattca

• Sequences
  • pKD13 Kanamycin cassette with FRT