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From 2012.igem.org

Contents 1 Re-plate plasmid-containing cultures 2 Make 10 mg/mL Neo stock solution for plates/liquid cultures 3 Make O/N cultures for Bs strains that arrived today 4 Make 5X Spizizen salts solution

Re-plate plasmid-containing cultures

• The Ec DH5α cells containing the plasmids pBAV1K-T5 and pIM1463 were re-plated from the original plates made (7/09/12)to obtain fresh colonies for O/N cultures. A non-fluorescent colony from the pBAV1K-T5 plate was chosen to reduce the chance of getting clones from late-stage cells expressing high (and toxic) levels of GFP.

Make 10 mg/mL Neo stock solution for plates/liquid cultures

• Weigh out 150 mg (149.65) of Neo powder from refrigerator
• Bring to 15 mL with dH20, mix gently to dissolve solids
• Filter sterilize solution
• Store at -20_o C
• Working concentration = 20 ug/mL

Make O/N cultures for Bs strains that arrived today

• The Bs strains PERM311 (168), PERM704 (ΔyfhQ::Spc), and PERM739 (ΔMutSL::Neo) came today in the mail from Dr. Pedraza-Reyes in Mexico via agar plates of questionable sterility.
• A single colony was approximated as closely as possible from each of the plates and transferred into 2 mL of LB broth with the appropriate antibiotic: 100 μg Spc/mL for PERM704, 20 μg Neo/mL for PERM739.
• Incubate the cultures overnight at 34^o C

Make 5X Spizizen salts solution

• The following salts were measured out and filled to 1 L with dH₂O
• The solution was mixed to dissolve the solids, then autoclaved

Compound	Molecular Formula	Amount in g (Actual)	Concentration
Potassium Phosphate, monobasic	K2HPO4	14.8 (14.88)	85 mM
Potassium Phosphate, dibasic	KH2PO4	5.4 (5.41)	40 mM
Ammonium Sulfate	(NH4)2SO4	2.0 (2.01)	15 mM
tri-Sodium Citrate Dihydrate	Na3C6H5O7	1.9 (1.90)	6 mM
Magnesium Sulfate Heptahydrate	MgSO4•7H2O	0.2 (0.201)	0.8 mM