TBE solution for minigels

For 5X stock solution:

  • 27g Tris base
  • 13.75g Boric acid
  • 10mL 0.5M EDTA
  • Water to 500mL

Mix vigorously with a stir bar. May take 30 minutes or more to dissolve. Heating helps.

Autoclave to prevent crystallization.

Agarose gel for minigels

For 1% agarose, 1X TBE gels (enough for 4 gels):

  • 1.3g agarose
  • 130mL 1X TBE

Microwave until agarose dissolves completely and aliquot ~30mL into 4 labeled falcon tubes. Refrigerate til use - microwave 90 seconds to re-melt.

Add 3.0uL Gel Red just before pouring gel.

Load: 2uL sample +1uL loading dye per well. (2uL ladder)

Run: ~30 minutes @ 100V

Agarose gel for gel purification

For 0.7% agarose, 1X TBE gel (enough for 1 gel):

  • 0.5g agarose
  • 75mL 1X TBE

Microwave until agarose dissolves completely.

Add 7uL Gel Red just before pouring gel.

Large combs hold: ~50uL

Small combs hold: ~25uL

Load: 25uL sample + 1uL loading dye or 50uL sample + 2uL loading dye. (5uL ladder)

Run: 2.5-3 hours @ 50V (start with fresh well buffer)

Agarose gel for midigels (12x14cm) for sex barcoding

For 3% agarose, 1X SB gels (enough for 1 gel):

  • 3.9g agarose
  • 130mL 1X SB buffer

Microwave at 20-30% power, swirling every 30-60sec until agarose dissolves completely. Allow to cool 2-3 minutes

Add 5.0uL Sybr Safe, swirl gently to thoroughly mix, and pour gel.

Load: 5uL sample (+1uL loading gel if not included in pcr mix) or ladder per well.

Run: ~40 minutes @ 125V

Ampicillin stock solution for TA cloning

For 100mg/mL solution:

  • 500mg Ampicillin sodium salt in
  • 5mL ddH2O

Store in refrigerator.

IPTG stock solution for TA cloning

For 100 mM solution:

  • 119.2 mg isopropyl β-D-thiogalactopyranoside (IPTG)
  • 5 ml ddH2O

Store in -20 freezer.

LB +Amp Agar plates for TA cloning with Qiagen EZ Competent Cells

For 250mL (10 plates):

Combine in autoclavable 500mL (or larger) bottle:

  • 6.25g LB Broth Miller
  • 3.75g agar (for 1.5% agar)
  • 250mL ddH2O

Mix 5-10 minutes with stir bar w/out heat. It won’t go into solution until autoclaved.

Autoclave on the “Liquid 121C 20min/30min exhaust” setting. Don’t seal bottle lid.

Allow to cool to <50C (cool enough to touch but not so cool it starts to solidify).

Add 250uL Ampicillin solution (100mg/mL) and swirl to mix. Avoid bubbles.

Sterilize a work area in the fume hood (to keep an updraft).

Pour ~25mL of the LB+amp agar solution into each dish (To “fill arrow” or half full). Swirl bubbles to the edge.

Place lid partially open facing away from the fume hood opening to cool and solidify.

Let the agar solidify completely before closing the lid. If excessive moisture accumulates, dry upside down for 30 minutes in 37C incubator.

Label with “LB+amp” and the date, and store in a plastic bag in the refrigerator until ready to use. Best within a week—ampicillin loses effectiveness over time.

Prior to use, for each plate mix:

  • 12.5uL IPTG solution (100mM)
  • 50uL X-gal solution (40mg/mL)

Mix well and pipette 62.5uL of the mixture onto each plate. Spread over the entire surface with a flame sterilized spreader. Let sit at least 30 minutes before use.

This recipe yields final concentrations recommended for Qiagen EZ Competent Cells:

  • 100ug/mL Ampicillin
  • 50uM IPTG
  • 80ug/mL X-gal

3M Sodium Acetate Stock for Qiagen Gel Extraction

  • 1.23g Sodium Acetate (m.w. = 82.03g/mol)
  • 5mL ddH2O

Shake to mix in Falcon tube. Store in refrigerator.

1M HCl from concentrated HCl

(37% w/w concentrated HCl is equivalent to ~12M)

  • 110mL Water
  • 10 mL concentrated 37% w/w HCl

Add the acid to the water—NOT the other way around.