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    • CommentAuthorjround77
    • CommentTimeMay 25th 2010 edited
     
    You will need:

    Bucket of ice
    100ng-500ng (~2 uL) of plasmid DNA to be transformed in a sterile eppendorf tube
    Competent bacteria cells (DO NOT THAW until ready to begin)
    Heat block set to 42C
    LB Broth
    Bunsen Burner
    Micropipettors and pipette tips
    37C water bath
    2 LB agar plates with appropriate antibiotic
    37C plate incubator

    NOTE: Plate only in the PM

    1. Turn on heat block to 42C
    2. Transfer ~1-2 ul of plasmid DNA to fresh eppendorff tube
    3. Allow competent cells to thaw on ice. Do not use your hands to speed it up. (NOTE: comp. cells are in -80C freeze; when thawed, they still appear cloudy, so must flick them; discard unused comp. cells)
    4. As soon as they are thawed, add 50-100uL of the cells to the tubes containing the plasmid DNA.
    5. Incubate on ice for 15'. With 10' left, check temp of heat plate (MUST be 42C)
    6. Heat-shock the cells by placing in the 42C heat block for 90 seconds.
    7. Return the tube to ice.
    8. Incubate on ice for 2'.
    9. Add 300uL of sterile LB broth to the tube using sterile technique. (expose lip of LB Broth bottle to flame from Bunsen Burner; keep bottle close to flame so bacteria don't get in)
    10. Incubate in a 37C water bath for 45'-1 hour.
    11. Plate an aliquot of the cells (~30ul) on LB/antibiotic plates. Use "hockey sticks" from 9" glass pipets to spread the cells around petri dish. (Use bunsen burner to make the hockey sticks)
    12. Invert plates and grow at 37C overnight (No more than 18-20 hours).

    AFTER OVERNIGHT INCUBATION:
    1. Aliquot 2ml of the LB+Amp solution (0.02g Amp/200ml LB) into 14ml round bottom tubes
    2. Using a yellow pipet tip, pick up an isolated large colony and drop the tip into the correct tube
    3. Cap and place into incubator (37C) with shaker set to 200rpm for ~6 hours

    Note on plating: If you are transforming a plasmid of known concentration, you will only need to plate 20-30uL. If concentration is unknown, plate 20uL and 120uL. If you are transforming a ligation reaction, you will want to plate more cells, generally around 100-120uL, on multiple plates.

    **Transformation of Zymo Research Competent Cells

    1. Thaw cells on ice.
    2. Combine DNA (100ng-500ng or 5ul of eluate from TE-soaked paper) with at least 50ul cells.
    3. Incubate on ice 5 minutes.
    4. Spread cells on plates, invert plates and incubate at 37C overnight.

    If transforming a plasmid with antibiotic resistance OTHER THAN ampicillin, re-suspend cells in 200ul of LB following the 5-minute incubation on ice. Incubate in 37C water bath for 45’-1 hour, then plate cells as above.