Carrico:Standard Protocols: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Juah Chung (talk | contribs) No edit summary |
Juah Chung (talk | contribs) No edit summary |
||
Line 1: | Line 1: | ||
{{Template:carrico}} | {{Template:carrico}} | ||
==DNA== | |||
===Ordering oligos through Stony Brook University=== | ===Ordering oligos through Stony Brook University=== | ||
Line 16: | Line 16: | ||
*Should terminate in one or more C or G bases | *Should terminate in one or more C or G bases | ||
*Melting temperature should be less than or equal to 78°C (Tm = 81.5 + 0.41 (%GC) – 675/N - % mismatch where N = total number of base pairs) | *Melting temperature should be less than or equal to 78°C (Tm = 81.5 + 0.41 (%GC) – 675/N - % mismatch where N = total number of base pairs) | ||
===Gel extracting DNA=== | |||
QIAquick Gel Extraction Kit Protocol (using a microcentrifuge) - [[:Media:QIAquick Gel Extraction Kit Protocol.pdf|pdf file]] | |||
===Determining DNA template concentration (and amount)=== | ===Determining DNA template concentration (and amount)=== |
Revision as of 10:28, 14 July 2009
Carrico Lab at Stony Brook
Home Lab Members Recipes Standard Protocols Publications Links
DNA
Ordering oligos through Stony Brook University
- Go to DNA Sequencing Facility
- Click on "Custom Oligos"
- Click on "Ordering" link at bottom of page
- Log-in
- Click on "Order"
Guidelines to determine primer sequence for Quikchange
- Between 25 to 45 base pairs
- Desired mutation should be in middle of primer with 10 to 15 bases of correct sequence on both sides
- Minimum GC content = 40%
- Should terminate in one or more C or G bases
- Melting temperature should be less than or equal to 78°C (Tm = 81.5 + 0.41 (%GC) – 675/N - % mismatch where N = total number of base pairs)
Gel extracting DNA
QIAquick Gel Extraction Kit Protocol (using a microcentrifuge) - pdf file
Determining DNA template concentration (and amount)
- Find absorbance (OD) by:
- In a micro cell, pipette 50uL water
- BLANK at 260nm (remember to turn both vis & UV lamps on and wait 5 minutes)
- Pipette out 5uL water
- Pipette in 5uL DNA template
- Place micro cell in spectrophotometer & READ (remember light pathway for placement)
- Clean micro cell by washing with 2mL water followed by 2mL methanol/ethanol from wash bottles
- Go to DNA Concentration Calculator
Competent Cells
Making electrocompetent cells
Preparation of materials (previous day)
- Sterilize 2x 500mL LB media in 2L flasks and 2L DI water
- Sterilize at least 50 Eppendorf tubes, 200uL pipette tips and 1000uL pipette tips
- Wash 4 large centrifuge bottles and sterilize with ethanol (do NOT use autoclave)
- Place dry capped bottles and sterilized water in Cold Room on 6th floor
- Sterile filter 10% glycerol solution and place in Cold Room on 6th floor
- Assemble the following:
- 200uL and 1000uL pipettors and sterile tips
- Pipetboy
- At least 10 disposable 10mL pipettes
- Large round dewar
- Sterile tubes
- 1 large Eppendorf tube rack
- 1 sterile 50mL Falcon tube
Preparation of cells
- Grow 2x 5mL starter cultures of the cells to be made competent overnight
- Inoculate 1L (2x 500mL in 2L flasks) LB broth with starter cultures
- Grow at 37C to an OD(600) of 0.5-0.8 (at this point, cells should be in log-phase growth)
- Transfer flasks to an ice bucket and chill on ice 15-30min
- Move to cold room
- Transfer cells to large centrifuge bottles
- Balance and centrifuge at 4C and 4000rpm (~4000g) for 12 minutes
- Immediately place bottles back on ice and (in cold room) empty supernatant
- Resuspend pellets in an equal volume of sterilized water using the Pipetboy and disposable pipettes
- Centrifuge at 4C and 4000rpm for 12 minutes
- Pour off supernatant
- Resuspend pellets in ~10mL sterile water each
- Consolidate from 4 bottles to 2 bottles
- Fill up bottles to ~350mL, balance and centrifuge
- Pour off supernatant
- Resuspend pellets in ~10mL cold sterile 10% glycerol solution and transfer to 50mL Falcon tube
- Balance a second Falcon tube with water
- Spin Falcon tubes at 4C and 7800 rpm (6500g) for 20min
- Resuspend to a final volume of ~2mL cold 10% glycerol
- Aliquot into Eppendof tubes (50uL each)
- Flash freeze in liquid Nitrogen bath
- Place aliquots in -80C freezer
Making chemically competent cells
- Start with 5uL cell (-80°C)
- Inoculate in 5mL 2XYT (Needs to be VERY STERILE!)
- Leave overnight in incubator at 37°C
- Mix 500uL from overnight & 50mL 2XYT using pipette boy
- Incubate at 37°C & 250rpm until OD readings of 0.600 (~2.5hr)
- Note: E. coli strains generally double after ½ hour
- Place on ice for 30 minutes
- While “on ice,” make CaCl2 (& glycerol) solutions
- 30mL 50mM CaCl2 (dilute from 1M CaCl2)
- 7mL 50mM CaCl2 & 20% glycerol
- Centrifuge at 4°C, 3000rpm for 15 minutes
- Resuspend in 25mL 50mM CaCl2 (makes cells permeable)
- Use pipette boy to be extra sterile
- Do NOT mix too much
- Place on ice for 60 minutes
- Centrifuge at 2000rpm for 10 minutes
- Resuspend in 5mL 50mM CaCl2 & 20% glycerol
- In Ice Room on 6th floor, aliquot 105uL into 600uL eppendorfs
- Flash freeze with liquid nitrogen in deuer
- Place tubes in a freezer box & store in -80°C freezer
Note: Check transfer efficiency using a known plasmid.
Vector NTI
Creating a new plasmid
- Open the Vector NTI Explorer program
- Click on the "New" icon
- Fill-in "Name"
- Under the "Sequence" tab, click on "Edit Sequence"
- Paste-in the sequence
- Select "OK"
- Double-click on newly created plasmid
Creating a gene in a plasmid
- Highlight the plasmid you would like edited
- Select "Edit" --> "New" --> "Add feature"
- Select the name of the plasmid
- Select "Save as"
Changing nucleotides
- Highlight bases to be changed
- Select "Edit" --> "New" --> "Replace"
- "Save As" a different name