Team:NYMU-Taipei/Notebook-Protocol

E.coli protocol

Two-day efficient cloning cycle

We used an efficient two day cloning cycle split into a "Light" day and a "Heavy" day.

Light Day

The light day consists of Colony PCR and liquid culture of colonies transformed from a previous day.

1. The 3-in-1

First, count the number of colonies you want to check. Then, do the following 3 things sequentially:

  • Liquid culture

  • 2nd time plate

  • Colony PCR

    • (Use the same tip to add the template to these three things)

    2. Make the gel for electrophoresis

    3. Run gel electrophoresis to check the colony PCR product


    Heavy Day


    The heavy day consists of:

  • Previously grown plasmid extraction

  • Plasmid PCR

  • Gel extraction

  • Digestion

  • Ligation

  • Transformation

    • Colony PCR (Thermo DreamTaq®)


  • Make the Colony PCR mix (we use Thermo' DreamTaq) with the mix amount slightly modified:

    • Item uL
    Primer(Forward and reverse) 1
    dNTP (10mM) 1
    10x DreamTaq buffer 5
    Taq Polymerase 0.2
    ddH20 42.8
    Total 50

  • Select a colony using a tip or toothpick.

  • Dip it in a PCR tube and swirl it around.


    • PCR run protocol

    Temperature Time  
    94℃ 60s  
    94℃ 15s  
    55℃ 20s 30-35 cycles
    72℃ 1kb/min + 5-10s  
    72℃ 300s  

    Liquid culture:

  • Aliquot 4 mL of LB medium in a centrifugal tube for each colony.

  • Use a tip and dip it in the LB culture and swirl it around.

    • Draw on 2nd time plate:

    Take out new plates from the fridge and divide them into smaller sections. This is the second-time plate. Make sure to write the date on it. Cross out in red pen any sections you know is wrong after the Colony PCR check

    Making Gel for gel electrophoresis

  • Make either 100mL or 150mL of gel at at time and select a relevant percentage (e.g. 1%, 1.5%, or 2%)

  • Measure out the relevant percentage in agarose (e.g. 1g of agarose for 100mL of 1% gel).

  • Fill it up with 1xTAE buffer.

  • Microwave on low, constantly taking it out every so often to swirl and mix it. Keep microwaving until clear. Make sure it is completely clear

  • Cool to a temperature that is still hot but still can be held in your hand. If the gel gets too cold and starts to harden, start microwaving again until clear.

  • Add 5uL of Safe-seeing dye for every 100mL of gel after cooled to the correct tempearature. Mix it well by swirling

  • Pour it onto the molds quickly. Put a cover on it to block out the light.

  • Wait at least 15-20 min until using it.

  • Store in 4°C and away from light.

    • Gel electrophoresis

  • Select a relevant % agarose gel based on your own experience

  • Load 5uL from each tube of Colony PCR, mix it with 1uL of 6x DNA Dye and put it in a well.

  • Load 3uL of marker into a well.

  • Run in the 13x13cm box at 60V or 70V and 400mA for the desired amount of time.

  • View in the gel viewer machine.

    • Plasmid extraction


    PCR (Dream Taq or KOD)

    Dream Taq:

    Item uL
    Primer(Forward and reverse) 0.2
    Template(100ng) ?
    dNTP (10mM) 0.2
    10x DreamTaq buffer 1
    Taq Polymerase 0.04
    ddH20 to 10 uL
    Total 10

    KOD:
    KOD是這種嗎?

    https://www.merckmillipore.com/TW/zh/product/KOD-Hot-Start-DNA-Polymerase,EMD_BIO-71086?ReferrerURL=https://www.google.com.tw/&bd=1


    Item uL
    Primer(Forward and reverse) 0.2
    Template(100ng) ?
    dNTP (10mM) 0.2
    10x KOD buffer 1
    KOD Polymerase 0.04
    Mg2+
    ddH20 to 10 uL
    Total 10

    PCR (Dream Taq or KOD)


    Digestion

    [Which enzymes we use???]

    Insert and backbone:

    Item
    DNA 600 or 1000ng
    10x Buffer 2uL
    Enzyme1 0.6 or 1 uL
    Enzyme2 0.6 or 1 uL
    ddH2O to 20 uL
    Total 20

    Backbone check:
    To check if the backbone is cut.

    Item
    DNA 100ng
    10x Buffer 1 uL
    Enzyme1 or 2 0.1 uL
    ddH2O to 10 uL
    Total 10

    Digest for at least 1hr. Over 2hr is better. EcoRI doesn't have star activity when cut this way (even overnight)

    We run insert digests and backbone digests with backbone check on a gel. Then use GeneAid's gel extraction kit. The modifications in the protocol include:

  • Warm EB to 60-70oC before elution.

  • Always use the Gel (sequencing) protocol for gel extraction.

    • Ligation

    We use Thermo's and NEB's T4 Ligase:

    Item amount
    Vector 8.5uL, total of approximately 100ng of DNA.
    Insert
    ddH2O
    10x Ligase Buffer 1
    T4 Ligase 0.5
    Total 10

    Incubate at room temperature for 2hr then transform 1uL then put the remaining amount in a small bag and put in 4oC overnight in case the transformation fails and retransformation is required.

    Transformation

    We majorly use commercial E. coli DH5α competent cells.

  • Add 1uL of plasmid or ligation mix to 20 uL of competent cells.

  • Put mixture on ice for 30 minutes.

  • Heat shock at 42℃ for 1 min.

  • Put the mixture back on ice for another 20 minutes.

  • Add 200 uL of LB broth to repair the cell wall; incubate at 37oC for 1.5 hr.

  • Plate it on a relevant antibiotic plate.

  • Incubate plate at 37oC overnight.

    • Objective: (1) Extract Hemolymph

    (2) Test the efficiency of Pmcl1 (With control groups: 1. Minimal medium

    1. Sterile ddH2O
    2. SDB broth)

    Methods:

    1. Hemolymph Extraction:

    Description

    Notes

    1.

    Leave斜紋夜盜蛾/蠶蛾 larvae buried in ice until they stop moving (3-10 min)

    2.

    Place larvae in 70% ethanol bath for 30 seconds then air dry them on 無塵紙

    3.

    Make an incision on the side of the last proleg and rest the incision site on the rim of the Eppendorf tube to collect the bluish hemolymph. (DO NOT SQUEEZE THE INSECT)

    Sterilize scissors/scalpel by dipping them in 70% ethanol solution and dry or burn with ethanol lamp (if burn wait till the scissors/scalpels cool before using)

    The larvae will not bleed if it becomes too cold so warm in room temperature when this happens

    Avoid collecting fat bodies (white tissues) and stop collection immediately if yellow tissue (Gut) is protruding from the incision site

    4.

    Put Eppendorf tubes in 60°C water bath for 30 min to prevent melanization

    5.

    Chill Eppendorf tubes on ice

    (disposal)

    6.

    Centrifuge the hemolymph tubes using the small centrifuge for 2 min

    7.

    Collect the supernatant (careful not to disturb the pellet) and move it to a new tube, label and store in -20°C freezer.

    Reference:

    1. http://www.acad.carleton.edu/curricular/Biol/resources/rlink/lab1p3.html
    2. Singh S, Reese JM, Casanova-Torres AM, Goodrich-Blair H, Forst S 2014.Microbial population dynamics in the hemolymph of Manduca sexta infected with Xenorhabdus nematophila and the entomopathogenic nematode Steinernema carpocapsae. Appl Environ Microbiol 80:4277–4285. doi:.10.1128/AEM.00768-14