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.
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
- 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 |
20X SSC (pH 7.0)
|
|
NaCl |
175.3 g/L |
sodium citrate |
88.2 g/L |
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 |
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 |
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