E. coli knockout

From CSBLwiki

Revision as of 07:47, 2 August 2010 (view source) Csbl (Talk | contribs) (→Procedure) ← Older edit		Revision as of 07:47, 2 August 2010 (view source) Csbl (Talk | contribs) (→Procedure) Newer edit →
Line 42:		Line 42:
	***Overview is attached.  [[media:overview.jpg|File:Overview]]		***Overview is attached.  [[media:overview.jpg|File:Overview]]
	*PCR condition		*PCR condition
-	**Anneling temparature and extension time is depend on the target sequence.	+	** Anneling temparature and extension time is depend on the target sequence.
-	**Anneling temparature is tested at 55℃ at first.	+	** Anneling temparature is tested at 55℃ at first.
-	**Extension time is depend on the length of the target sequence. (1kb = about 1 min.)	+	** Extension time is depend on the length of the target sequence. (1kb = about 1 min.)
-	[[Image:PCR condition.jpg|center|thumb|100px]]	+	** Detail conditions are attached.[[Image:PCR condition.jpg|center|thumb|400px]]
	==== Notes ====		==== Notes ====
	*Primers		*Primers

---

Revision as of 07:47, 2 August 2010

Contents 1 E. coli Gene Replacement 1.1 Lambda red recombinase method 1.1.1 Materials 1.1.2 Procedure 1.1.3 Notes

E. coli Gene Replacement

Lambda red recombinase method

(see this link - openwetware::gene replacement - for the method comparison)

Materials

• Plasmids
  • pKD46 -sequence info.
    • Coding lambda red recombinase
    • Ampiciline resistance
    • Temperature sensitive replication origin
  • pKD13 - sequence info.
    • 3.4kb, KanR flanking FRT sequence, R6K gamma replication origin
    • Kanamycin resistance gene template plasmid
  • pCP20
    • Flipase system
    • Ampiciline resistance
    • Temperature sensitive replication origin
• 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

• 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.
    • Overview is attached. File:Overview
• PCR condition
  • Anneling temparature and extension time is depend 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.)
  • Detail conditions are attached.
    

    

Notes

• Primers