446/546 handout 21nd Aug. Making solutions AND homework.
Several solutions need to be prepared for downstream experiments involving nucleic acid
extraction, gel electrophoresis and DNA sequencing.
GENERAL ADVICE, make solutions in beakers if you need to add large amounts of chemicals or
make large volumes. Tubes, bottles, cylinders may be narrow and limit ease of adding chemicals.
So think before you act. Wear gloves, coat when handling chemicals.
CLEAN SPATULA’S ETC. DO NOT CONTAMINATE CHEMICALS
INSTRUCTIONS:
1) YOUR GROUP WILL MAKE SOLUTION NUMBER 4 and ___
2) Locate the necessary ingredients (chemicals, stock solutions, water) and the appropriate
hardware (cylinder, beaker, bottle or reaction tube, scale, weighing boats/paper, stirring rods,
mixer, pH meter, hood). You may have to share resources/wait your turn
3) From the recipe, calculate amounts (weights and/or volumes) needed for each ingredient and
have your numbers checked by an instructor BEFORE proceeding.
4) Measure/weigh out the required amounts of the chemicals and combine these to make your
solutions. GO TO THE HOOD IF THIS NEEDED. IMPORTANT, COMBINE ALL
INGREDIENTS INTO A VOLUME THAT IS NO MORE THAN 80% OF THE FINAL
VOLUME . ONLY AFTER ALL CHEMICALS ARE ADDED, DISSOLVED AND pH IS
CORRECT (If needed), TRANSFER TO CYLINDER AND ADD WATER TO THE FINAL
VOLUME!
5) Clean glassware afterwards!!
Solutions:
1) CTAB (DNA extraction buffer)
Make 100 ml final volume (start with 80% of final volume) with the following ingredients
2% (w/v) CTAB
1.4M NaCl
20 mM EDTA (disodium-dihydrate)
100 mM Tris/HCL (from stock solution)
0.2% (v/v) beta mercapto ethanol (TOXIC/SMELLS BAD USE THE HOOD)
Check/adjust pH 8.0, add dH2O to final volume
CTAB dilution may require warming to 60C, before use (NOT NOW), add 1/100 volume of
proteinase K stock (10mg/ml dH2O)
2) Ammonium acetate/Ethanol (DNA clean-up)
Make 100 ml final volume (start with 80% of final volume) containing
76% (v/v) EtOH
10 mM NH4Ac
dissolve, add dH2O to final volume
3) 50x TAE, (Tris Acetate EDTA) for gel electrophoresis
Make 100ml final volume (start with 80% of final volume) containing
2 M Tris base
50 mM EDTA (from 0.5 M stock, pH 8.1)
2 M Glacial Acetic Acid (CORROSIVE ACID, HOOD from 17.5 M stock)
check/adjust pH 8.0
add dH2O to final volume
4) electrophoresis layer buffer (sample buffer, blue juice)
Make 10 ml final volume (start with 80% of final volume) containing
50% (v/v) glycerol
100 mM EDTA (from 0.5 M EDTA, pH 8.1, stock)
0.05% (w/v) SDS (from 10% (w/v) SDS stock)
0.1 % (w/v) Bromophenol Blue (DO NOT SPILL ON CLOTHES, PERSISTENT STAIN)
dissolve, add dH2O to final volume
5) 0.25 M EDTA stock
Make 100 ml final volume (start with 60% of final volume) containing
0.25 M EDTA
pH 8.1 (use NaOH)
dissolve (note EDTA only dissolves if pH>8.0), add dH2O to final volume
6) 3M NaAc, pH 5.2 (DNA sequencing)
Make 100 ml final volume (start with 80%) containing
3M Sodium Acetate
pH 5.2 (adjust with HCl or NaOH)
dissolve, add dH2O to final volume
Homework, answer in your note book
Provide answers to questions 1-3 in the format of x ml EtOH stock + y ml water
1) How much volume of 100% EtOH is needed for 100 ml of a 70% EtOH dilution?
2) How much volume of 95% EtOH is needed for 95 ml of a 70% EtOH dilution?
3) How much volume of 100% EtOH is needed for 50 ml of a 80% EtOH dilution?
4) You are preparing a 0.5 M EDTA solution, the EDTA powder does not dissolve. How do you solve
this problem?
5) You have 40nmoles of a PCR primer, how much water (in ml) do you add to make a 50 micromolar
stock solution?
6) You have just finished making 1 L of a 50x stock solution for a technique that you and all your 20
colleagues in the lab perform almost every day. You realize that you forgot to adjust the pH. What do
you do?