Carrico:Standard Protocols

Carrico Lab at Stony Brook

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DNA

Ordering oligos through Stony Brook University

1. Go to DNA Sequencing Facility
2. Click on "Custom Oligos"
3. Click on "Ordering" link at bottom of page
4. Log-in
5. 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)

1. Find absorbance (OD) by:
   1. In a micro cell, pipette 50uL water
   2. BLANK at 260nm (remember to turn both vis & UV lamps on and wait 5 minutes)
   3. Pipette out 5uL water
   4. Pipette in 5uL DNA template
   5. Place micro cell in spectrophotometer & READ (remember light pathway for placement)
2. Clean micro cell by washing with 2mL water followed by 2mL methanol/ethanol from wash bottles
3. Go to DNA Concentration Calculator

Purifying DNA

QIAprep Miniprep Protocol - pdf file

Protein

Site Specific Incorporation of a Fluorinated Amino Acid in vivo (21st Pair)

Site Specific Incorporation of a Fluorinated Amino Acid in vivo Protocol - pdf file

Competent Cells

Making electrocompetent cells

Preparation of materials (previous day)

1. Sterilize 2x 500mL LB media in 2L flasks and 2L DI water
2. Sterilize at least 50 Eppendorf tubes, 200uL pipette tips and 1000uL pipette tips
3. Wash 4 large centrifuge bottles and sterilize with ethanol (do NOT use autoclave)
4. Place dry capped bottles and sterilized water in Cold Room on 6th floor
5. Sterile filter 10% glycerol solution and place in Cold Room on 6th floor
6. 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

1. Grow 2x 5mL starter cultures of the cells to be made competent overnight
2. Inoculate 1L (2x 500mL in 2L flasks) LB broth with starter cultures
3. Grow at 37C to an OD(600) of 0.5-0.8 (at this point, cells should be in log-phase growth)
4. Transfer flasks to an ice bucket and chill on ice 15-30min
5. Move to cold room
6. Transfer cells to large centrifuge bottles
7. Balance and centrifuge at 4C and 4000rpm (~4000g) for 12 minutes
8. Immediately place bottles back on ice and (in cold room) empty supernatant
9. Resuspend pellets in an equal volume of sterilized water using the Pipetboy and disposable pipettes
10. Centrifuge at 4C and 4000rpm for 12 minutes
11. Pour off supernatant
12. Resuspend pellets in ~10mL sterile water each
13. Consolidate from 4 bottles to 2 bottles
14. Fill up bottles to ~350mL, balance and centrifuge
15. Pour off supernatant
16. Resuspend pellets in ~10mL cold sterile 10% glycerol solution and transfer to 50mL Falcon tube
17. Balance a second Falcon tube with water
18. Spin Falcon tubes at 4C and 7800 rpm (6500g) for 20min
19. Resuspend to a final volume of ~2mL cold 10% glycerol
20. Aliquot into Eppendof tubes (50uL each)
21. Flash freeze in liquid Nitrogen bath
22. Place aliquots in -80C freezer

Making chemically competent cells

1. Start with 5uL cell (-80°C)
2. Inoculate in 5mL 2XYT (Needs to be VERY STERILE!)
3. Leave overnight in incubator at 37°C
4. Mix 500uL from overnight & 50mL 2XYT using pipette boy
5. Incubate at 37°C & 250rpm until OD readings of 0.600 (~2.5hr)
   • Note: E. coli strains generally double after ½ hour
6. Place on ice for 30 minutes
   • While “on ice,” make CaCl2 (& glycerol) solutions
   1. 30mL 50mM CaCl2 (dilute from 1M CaCl2)
   2. 7mL 50mM CaCl2 & 20% glycerol
7. Centrifuge at 4°C, 3000rpm for 15 minutes
8. Resuspend in 25mL 50mM CaCl2 (makes cells permeable)
   • Use pipette boy to be extra sterile
   • Do NOT mix too much
9. Place on ice for 60 minutes
10. Centrifuge at 2000rpm for 10 minutes
11. Resuspend in 5mL 50mM CaCl2 & 20% glycerol
12. In Ice Room on 6th floor, aliquot 105uL into 600uL eppendorfs
13. Flash freeze with liquid nitrogen in deuer
14. 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

1. Open the Vector NTI Explorer program
2. Click on the "New" icon
3. Fill-in "Name"
4. Under the "Sequence" tab, click on "Edit Sequence"
5. Paste-in the sequence
6. Select "OK"
7. Double-click on newly created plasmid

Creating a gene in a plasmid

1. Highlight the plasmid you would like edited
2. Select "Edit" --> "New" --> "Add feature"
3. Select the name of the plasmid
4. Select "Save as"

Changing nucleotides

1. Highlight bases to be changed
2. Select "Edit" --> "New" --> "Replace"
3. "Save As" a different name