- 西亚试剂：：MinElute DNA纯化技术
- 西亚试剂：：Oligonucleotide Purification
Last modified 10/16/98
Purpose: Purification of oligonucleotides (which have already been purified by reverse phase) can increase the efficiency of site directed mutagenesis from around 30% to ~75% or greater in some cases.
1. Use PAGE to purify 20-30 micrograms of oligonucleotide on a small 10% denaturing acrylamide urea gel (1 mm thick small gel). To do this, mix equal volumes of oligonu...详细+
- 西亚试剂：：Recovering DNA from agarose gels
Recovering DNA from agarose gels
Paul N. Hengen, Ph.D. (July 14, 1999)
Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the internet. A commonly occurring theme on the net is the recovery of DNA, and this month's column discusses the pros and cons of various methods used to extract DNA fragments directly from agarose gels....详细+
- 西亚试剂：：DNA Fragment Purification
10 mM Tris (pH to 7.5)
1 mM EDTA (pH to 8.0 to dissolve)
Frozen agarose gel piece containing the desired DNA fragment
Micropipetter and tips
Microcentrifuge and tubes
PCR machine and the 600-ul tubes for use in the machine
Ultrafree-MC(R) filter units, 0.45 um
Quick thaw the gel piece in a 600-ul tube using the PCR machine at 37 degrees C
Macerate the gel piece with a spat...详细+
- 西亚试剂：：Isolating DNA Fragments
Isolating DNA Fragments
• 0.8 % agarose gel in 1x TAE
• Digested DNA
• Glass Milk
• NaI solution
• New Wash
1) Run digested DNA out on agarose gel slowly (70 V on BioRad gel)
2) Use long wave UV lamp to visualize bands. Cut out band with scalpel. Cut smallest possible piece.
3) Put gel slice in an eppendorf tube and weigh to figure out volume of gel slice. (empty tube approx. 1 g)....详细+
- 西亚试剂：：DNA Plasmid Miniprep Protocol
DNA Plasmid Miniprep Protocol
1. Pick single colony and inoculate 5 ml of LB broth containing 200 g/l ampicillin or 1mg/5ml. Optional: Use a 15ml conical tube with a loosened cap and a piece of tape to hold it in place. Shake at 250 RPM 37oC overnight.
2. Centrifuge 1.5mL cells in 1.5 mL Eppendorf tube at top speed for 1 minute. Aspirate supernatant.
3. Resuspend cell pellet in 100 ul of GTE buffer (50 mM Glucose, 25 mM T...详细+
1. Spin down 1.5 ml of overnight culture in eppie for 1 minute on high.
2. Asprirate supernatant and resuspend cell pellet in 100 µl Solution I (25 mM Tris-HCl, pH 8.0, 10 mM EDTA).
3. Add 200 µl Solution II (0.2 N NaOH, 1.0% SDS) and mix gently by inversion.
4. Add 150 µl Solution III (3M KOAc, pH 4.8 [60 ml 5M KOAc, 11.5 ml HOAc, 28.5 ml H2O]), vortex briefly to mix, and spin for 5 minu...详细+
- 西亚试剂：：Boiling lysis mini-prep
Boiling lysis mini-prep
1. Grow an overnight culture from a single colony in 2 mls LB + antibiotic
2. Transfer 1.5 mls of culture to eppendorf tube. Spin for 1 minute on high and remove supernatant.
3. Add 700 µl STET. Add 25 µl lysozyme stock solution. Vortex to resuspend pellet. Place tubes on ice for 5-10 minutes.
4. Boil tubes for 1 minute.
5. Spin in microfuge for 10 minutes.
6. Pull out sno...详细+
- 西亚试剂：：DNA Plasmid Prep.
DNA Plasmid Prep.
This is a scaled up version of the Baron protocol which has been modified to achieve purity comparable to CsCl preps. I have not sorted out strain variations yet but HB101 works well.
1% glucose 10 g glucose
25 mM Tris pH 8.0 25 ml 1M Tris pH 8.0
10 mM EDTA 20 ml 0.5 M EDTA pH 8.0
up to 1 liter with Q
store at 4o
1% SDS 2.5 ml 20% SDS
0.2N NaOH 1.0 ml 10N N...详细+
- 西亚试剂：：MINI PREP BY BOILING
Pellet 1.5 ml of an overnight culture at 12,000 rpm in Eppendorf centrifuge at RT for 3 min.
Resuspend bacterial pellet in 350 µl of STET buffer.
Add 25 µl of freshly prepared solution of lysozyme (10 mg/ml in 10 mM Tris-Cl, pH 8).
Mix by vortexing for 3 sec.
Place the tube in a boiling water bath for exactly 40 sec.
Centrifuge the lysate at 12,000 g for 10 min at room temp.
Remove the glob of bacterial d...详细+