Carrico:Standard Protocols

Carrico Lab at Stony Brook

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QIAquick Gel Extraction Kit Protocol (using a microcentrifuge) - pdf file

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)

How to determine 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

"How to" for Vector NTI

How to create 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

How to create 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"

How to change nucleotides

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