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Miscellaneous Molecular Biology Methods

EcoEvoDevo Lab

Updated 7 July 2022

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By Victuallers - Own work, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=35119980

Measuring concentration and DNA storage

  • Use the NanoDrop spec to measure the concentration of DNA. Record this information in your notebook and on the side of the tube. Also note the purity of the sample. Pure DNA should have an A260/A280 ratio ~ 1.8 and an A260/A230 ratio > 1.8
  • Store DNA at 4˚C for less than a week or at -20˚C for longer periods.

PCR purification

Many methods exist for the purification of DNA produced in PCR from used Taq and unincorporated primers and dNTPs. Here two methods are described.

Standard QiaQuick PCR purification

If a PCR produces a single band 100 bp to 10 kb, it can be purified using the QIAquick PCR Purification Kit (QIAgen catalog number 28104).

PEG Purification

This method recovers DNA over about 300 bp. Residual PEG should not inhibit most enzymatic reactions. This is the recommended purification method for PCR products used in gateway cloning. It's unclear whether it will be suitable for all applications.

  • Add water to your PCR product to bring it to 50 μl total volume.
  • Add 150 μl of TE Buffer (pH 8.0; 1X).
  • Add 100 μl of 30% PEG-8000, 30 mM MgCl2.
  • Vortex to mix thoroughly.
  • Immediately centrifuge the tube at 12,000 ×g for 20 minutes.
  • Carefully remove and discard the supernatant. The pellet will be clear and nearly invisible. If you remove about ¾ of the solution, then tip the tube, you should see an area at the bottom that looks gelatinous—that’s the pellet containing the DNA. Remove any remaining supernatant, but be careful to keep the pellet.
  • Dissolve the pellet in 30 μl of TE Buffer.

Phenol-Chloroform Extraction of Nucleic Acids

A 25:24:1 phenol:chloroform:isoamyl alcohol mix will avoid problems in purifying RNA and tighten the interface. Also, but sure the phenol is equilibrated to pH 7.8-8.0. It’s a good idea to do centrifugation steps at 4˚C, especially for RNA. If phases fail to separate, spin longer and harder! If worried about recovery, save the supernatant and organic phase, just in case.

  • Add 1 volume of phenol:chloroform and mix vigorously
    Spin for 5 min at maximum speed
    Save the aqueous, upper phase
  • Repeat this until no protein is visible at the interface.

If you’re worried about recovering maximum DNA, “back-extract”: Add some water to the used organic phase, mix, spin, and add the aqueous layer to the new step.

  • Add an equal volume of chloroform and mix vigorously, then let it sit for ~3 min
  • Spin for 5 min at maximum speed. Save the aqueous, upper phase.
  • From here it’s a standard NH4OAc precipitation…

Precipitation of Nucleic Acids with Ammonium Acetate and Ethanol

This is a modified version of the classic protocol for DNA and dsRNA.

  • Add 0.5 volume of cold 7.5M ammonium acetate and 2 volumes of cold 100% ethanol and vortex
  • Incubate at -20˚C for 20 min (longer incubation gives better yield, as long as 48 h)
  • Spin for 20 min at 12,000 rpm (for RNA use a refrigerated centrifuge at 4˚C)
  • Discard the supernatant
  • Wash with 1ml cold 70% ethanol
  • Spin for 5 min at 10,000 rpm
  • Discard the supernatant
  • Dry the pellet in a vacuum centrifuge (speed-vac). Try not to completely desiccate it. It should be translucent, not an opaque white.
  • Resuspend in water as needed

Alkaline Hydrolysis of RNA Probes

Prepare reagents fresh.

  • Prepare 200 mM sodium carbonate (Add 21.2 mg Na2CO3 to 1ml H2O)
  • Prepare 200 mM sodium bicarbonate (Add 16.8 mg NaHCO3 to 1ml H2O)
  • To 50 ul of resuspended, cleaned probe, add 30 ul of sodium carbonate and 20 ul of sodium bicarbonate
  • Incubate at 60ºC for the appropriate time
    To find the appropriate time (t), use the equation: t = (Lo-Lf)/(KLoLf), where Lo and Lf are the original and final probe lengths in kb, and K is 0.11 min-1 kb-1. For a 1-kb probe, hydrolysis to 200bp should take about 35 min
    • Move the tubes to ice, then spin briefly.
    • Neutralize by adding 3 ul 3M NaOAc and 5 ul 10% glacial acetic acid
    • Add 0.1 volume of 4M LiCl and 3 volumes of cold 100% ethanol.
    • Incubate at -20ºC for 30 min (to overnight)
    • Spin for 20 min at maximum speed
    Discard the supe
    • Speed-vac the pellet
    • Resuspend as needed

Preparation of E. coli freezer stocks

  • Add 850 ul of glycerol to a screw-top 2-ml freezer tube
  • Add 150 ul of bacterial culture (grown overnight) and stir well with the pipette tip, then vortex.
  • Store indefinitely at -80˚C
  • To recover the stock, scrape the frozen culture with a sterile inoculating loop and immediately streak onto LB agar containing the appropriate antibiotics. Incubate plates overnight at 37ºC.

Stock solutions

LB Broth / Plates

water ~800 ml
Bacto Tryptone 10 g
Bacto yeast extract 5 g
NaOH 1 pellet
NaCl 10 g
water (to 1 L)
agar (for plates) 15 g
  • Stir until mixed
  • Autoclave
  • Before adding antibiotics, cool to <50˚C on an orbital mixer
  • Pour plates by lifting the Petri dish lids minimally
  • Allow plates to set and cool at room temperature (Cover them if they contain antibiotics, which are light sensitive)
  • Store in a plastic sleeve at 4˚C

Antibiotic Stocks

[stock] amount [final]
Kanamycin 50 mg/ml 1 ml/L 50 ug/ml

DEPC Water

DEPC 4 ml
Water 4 L
  • Cover with foil
  • Stir overnight
  • Aliquot
  • Autoclave

5M NaCl

[final]
NaCl 292.25 g/L 5 M

10N NaOH

amount [final]
NaOH pellets 200 g 10 N
water 500 ml
  • Stir until dissolved
  • Store at room temperature in plastic bottle (not glass)

10X PBS (pH 7.4)

NaH2PO4 2.56 g/L
Na2HPO4 11.94 g/L
NaCl 102.2 g/L
  • Autoclave.

20X SSC (pH 7.0)

NaCl 175.3 g/L
sodium citrate 88.2 g/L
  • Autoclave.

1M Tris (pH 7.5)

[final]
Tris base 121.14 g/L 1 M
  • Add HCl to pH 7.5
  • Autoclave

1X TBE

Tris 10.8 g/L
boric acid 5.5 g/L
EDTA 0.93/L
  • Autoclave.

DAPI Stain

DAPI 10 mg
water up to 10 ml
  • Aliquot 100 ul, add 900 ul water, store foil-wrapped at 4C.

DNA Ladder

[stock] volume
1kb ladder 50 ul
loading dye 6X 83.3 ul
NaCl 4M 2.5 ul
TE buffer 1X 364.2 ul
  • Run 4-5ul on a gel

Denhardt’s Solution

ficoll 5 g
polyvinylpyrrolidone 5 g
bovine serum albumin (fraction V) 5 g
water to 500 ml
  • Stir covered until dissolved (~3h)
  • Aliquot
  • Store at -20C

Oncopeltus In Situ Hybe

[stock] volume [final]
foramide 25ml 50%
SSC 20X 12.5ml 5X
heparin (Sigma H-7005) 100mg/ml 50ul 100ug/ml
calf thymus DNA (Sigma D-8661) 10mg/ml 0.5ml 100ug/ml
yeast RNA 2.5mg/ml 2ml 100ug/ml
Tween-20 50ul 0.1%
water (to 50ml) 8.5ml

Yoshi's In Situ Hybe | [stock] | V | [final]

[stock] volume [final]
foramide 25ml 50%
SSC 20X 12.5ml 5X
heparin (Sigma H-7005) 50mg/ml 100ul 100ug/ml
yeast RNA 50 mg/ml 100 ul 100ug/ml
CHAPS 10% 0.5 ml 0.1%
Denhardt’s solution 50X 1 ml 1X
Tween-20 10% 0.5 ml 0.1%
water (to 50ml) 10.8 ml

Candice's In Situ Hybe

[stock] volume [final]
foramide 25ml 50%
SSC 20X 12.5ml 5X
salmon/herring sperm DNA (boiled 1 min) 40 mg/ml 125 ul 100ug/ml
Denhardt’s solution 50X 1 ml 1X
Tween-20 10% 0.5 ml 0.1%
water (to 50ml) 10.8 ml

Simple Hybe Solution

[stock] volume [final]
foramide 25ml 50%
SSC 20X 12.5ml 5X
Tween-20 10% 0.5 ml 0.1%
water (to 50ml) 10.8 ml

Maleic acid buffer (pH 7.5)

maleic acid 11.61g
NaCl 8.77g
water to 1Ll)

Antibody Block with BSA

[stock] amount [final]
BSA (fraction V) powder 100 mg 2%
PBTw 1X to 50ml

Antibody Block with Boehringer Mannheim Blocking Reagent

[stock] amount [final]
BM blocking reagent powder 250 mg 5%
Triton X-100 10% 1.0 ml 0.2%
maleic acid buffer 1X to 50ml

Alkaline Phosphatase (AP) reaction buffer

[stock] amount [final]
Tris (pH 9.5) 1M 5ml 100mM
MgCl2 1M 2.5ml 50mM
NaCl 5M 1ml 100mM
Tween-20 50ul 0.1%
water (to 50ml) 41.5ml

EcoEvoDevo Lab