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B.1 Antibiotic stocks
B.1.1 Norfloxacin / Ciprofloxacin
| Reagent | Amount per 200 ml |
| 5 N NaOH | 400 ml |
| nor or cipro | 0.25 g |
This stuff is hard to get into solution. The NaOH is supposed to help. Stock is good at least 1 month in at 4
° C. Freezing for longer storage seems to be ok (according to Jamey; I haven't tried yet). Takes
very little of this stuff to tear up some bacteria. MIC is around 150 ng/ml. That is only 6
ml of stock solution in 50 ml. I use 75 ng /ml to cause DNA damage but not kill them (does slow them down a little).
B.2 Common Buffers
B.2.1 Phosphate Buffered Saline (PBS)
| Reagent | Amount per Liter |
| KCl | 0.2g |
| NaCl | 8g |
| KH2PO4 | 0.240g |
| Na2HPO4 | 1.44g |
Adjust pH to 7.4 with HCl, I always check with the pH meter and it never has needed adjusting. Autoclave to sterilize.
B.3 Agarose gel buffers what?
B.3.1 Tris-Acetate-EDTA (TAE)
I use the 4 L 50X TAE dispensor from Fisher.
Use 5-8 V/cm (higher V for short fragments, lower V for long ones)
B.3.2 Tris-Borate-EDTA (TBE)
I use the 4 L 10X TAE dispensor from Fisher.
Use 5-35 V/cm (higher V for short fragments, lower V for long ones)
B.3.3 Sodium Boric Acid (SB)
This is a buffer for running very fast gels (10x faster than TAE) at high voltage. Works best for shorter fragments (less than 2kb). Doesn't work as well with samples containing higher salt concentrations (e.g. restriction digests). More info can be found in
Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis..
| Reagent | Amount per Liter |
| NaOH | 8 g |
| Boric Acid | 45 g |
Fill to 1 L with H
2O .
Use 5-10 V/cm (higher V for short fragments, lower V for long ones)
B.4 DNA and RNA storage buffers
B.4.1 Annealing of two oligos
Use STE Buffer (10 mM Tris pH 8, 50 mM NaCl, 1 mM EDTA). The salt allows the oligos to anneal.
B.4.2 Resuspension of lypholized primers
I follow the FAQ from IDT: Dissolve the stock oligo in TE. Make the freezer stock at 100uM by adding a volume of TE ten times the number of nanomoles of DNA present in the tube (it is noted on the spec sheet provided by IDT.
For jobs requiring 24 or more primer pairs I order them in plates frozen in TE at 100uM concentration. I put the pairs on consecutive rows to ease the construction of primer pairs.
I typically aliquot to a stock at a concentration of 2uM. And use 1.5
ml in a 20
ml PCR.
B.5 Midiprep Solutions
Added: Sun May 28 18:41:34 EDT 2006.
Last Update: Sun May 28 18:41:34 EDT 2006.
B.5.1 Alkaline Lysis Solution 1
Just a little something to resuspend the guys in. I add the RNAse Cocktail fresh right before using. 1
ml of RNAse cocktail per 200
ml of Alkaline Lysis Solution 1. Store at 4
° C.
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| Sucrose | 50 mM | - | 0.45 g |
| Tris-HCl [pH 8.0] | 25 mM Tris | 1M | 1250 ml |
| EDTA | 10mM | 0.5M | 1 ml |
B.5.2 Alkaline Lysis Solution 2
This stuff lyses the cells.
| Reagent | Amount | Stock Conc. | Amount for 1 ml |
| 0.2 N NaOH | 0.2 N | 1 N | 200 ml |
| SDS | 1% | 20% | 50 ml |
B.5.3 Alkaline Lysis Solution 3
This neutralizes the lysate.
| Reagent | Amount | Stock Conc. | Amount for 50 ml |
| potassium acetate | 3 M | 5 M | 30 ml |
| glacial acetic acid | - | - | 5.75 ml |
B.6 ChIP Recipes
B.6.1 Palsson lysis buffer
Palsson lab adapted from Grossman protocol.
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| Tris-HCl | 10mM Tris | 1M | 500 ml |
| NaCl | 50mM | 5M | 500 ml |
| EDTA | 10mM | 0.5M | 1 ml |
| Sucrose | 20% | - | 10 g |
| Ready-lyse lysozyme | 0.2ml per 500 add fresh | - | |
B.6.2 Palsson 2x IP buffer
Palsson lab adapted from Grossman protocol.
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| Tris-HCl | 200mM Tris | 1M | 10ml |
| NaCl | 600mM | 5M | 6ml |
| Triton X-100 | 4% | - | 2ml |
| 1mM PMSF | added just prior to use | 100mM | 500 ml |
B.6.3 Dilution Buffer
Taken from www.abcam.com
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| Triton X-100 | 1% | - | 500 ml |
| EDTA | 2 mM | 0.5 M | 200 ml |
| NaCl | 150 mM | 5 M | 1.5 ml |
| Tris-HCl pH 8 | 20 mM | 1 M | 1 ml |
| PMSF | 1 mM added just prior to use | 100 mM | 500 ml |
B.6.4 Low Salt Immune Complex Wash Buffer
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| SDS | 0.1% | - | 250 ml (of 20%) |
| Triton X-100 | 1% | - | 500 ml |
| EDTA | 2 mM | 0.5 M | 200 ml |
| Tris-HCl pH 8 | 20 mM | 1 M | 1 ml |
| NaCl | 150 mM | 5 M | 1.5 ml |
B.6.5 High Salt Immune Complex Wash Buffer
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| SDS | 0.1% | - | 250 ml (of 20%) |
| Triton X-100 | 1% | - | 500 ml |
| EDTA | 2 mM | 0.5 M | 200 ml |
| Tris-HCl pH 8 | 20 mM | 1 M | 1 ml |
| NaCl | 500 mM | 5 M | 5 ml |
B.6.6 LiCl Immune Complex Wash Buffer
| Reagent | Amount | Stock Conc. | Amount for 50ml |
| LiCi | 0.25 M | 5 M | 2.5 ml |
| NP40 | 1% | - | 500 ml |
| deoxycholate | 1% | solid powder | 500 mg |
| EDTA | 1 mM | 0.5 M | 100 ml |
| Tris-HCl pH 8 | 10 mM | 1 M | 500 ml |