Team:Brown/Notebook/July1

From 2010.igem.org

Thursday, July 1 2010

Results of test streaking plates:

(Note: next time use plating beads; don’t just add cells to the center of the plate and stick directly into the incubator)

Null plate: looked like a lawn of normal e. coli. Amp+ plate (standard concentration of amp): Some e.coli with fuzzy fungus-looking stuff on it; a bit less dense.

We still need to find out if our competent cells (the XL1-Blue stock that is at -80°C) are contaminated or not.

So, we plated our “stock” XL1-Blues that we made on:

  • Gary Amp+
  • Gary Tet+
  • iGEM Chlor+

Incubated at 37°C overnight.


Double Digest of pNoTat and WillRS (two double digests) with BamHI and NcoI

We are doing this because we need sticky ends on WillRS and pNoTat. Then we will ligate WillRS into pNoTat.

20 µl double digest

  • 7.8 µl dH2O
  • 2.0 µl 10X NEB Buffer 3
  • 8.0 µl DNA (pNoTat)
  • 1.0 µl NcoI
  • 1.0 µl BamHI
  • 0.2 µl BSA

20 µl total volume

1 hour at 37°C. Started at 12:55 PM.


Purification of above digests

QIAquick PCR Purification Kit Protocol using a microcentrifuge (Manual page 19).

“Fragments ranging from 100bp to 10kb are purified from primers, nucleotides, polymerases, and salts using QIA quickspin columns in a microcentrifuge.”

  1. Add 5 volumes of Buffer PBI to 1 volume of the PCR sample and mix.
    • 20 µl original volume added 100 µl PB1
  2. Make sure it’s yellow (indicates correct pH).
  3. Place a QIAquickspin column in a provided 2ml collection tube.
  4. To bind DNA, apply the sample to the QIAquick column, for 30-60s.
  5. Discard flow-through.
  6. Add 0.75 L buffer PE to QIAquick column and centrifuge ~60s to wash.
  7. Discard flow through and place QIAquick column back in the same tube. Centrifuge for an additional 1 minute. You have to do this step. Otherwise residual EtOH will not be removed and that will mess things up.
  8. Place column in 1.5 mL microfuge tube.
  9. Added 30 µl buffer EB and let it stand for 1 minute to maximize DNA concentration. Make sure you add EB to the center of the column. Buffer EB is preferred because dH2O is not always at the correct/optimal pH and that may reduce elution efficiency.

Ligation of purified digest products

  • 7.5 µl 2X DNA ligase buffer (rapid)
  • 1 µl T4 DNA ligase
  • 0.5 µl vector (pNoTat)
  • 1.5 µl insert (WillRS)
  • 4.5 µl dH2O

15 µl total volume

1 to 2’ RT.

Control ligations:

  1. no insert
  2. no vector

Making CaCl2 competent XL1 Cells

Received stock of XL1 Blues from Gary

  1. Placed in incubator from ~12PM to 2 PM
  2. Cells on ice for ~20 minutes; placed in 6 culture tubes of around 400 µl each.
  3. Centrifuged at 3000 RPM for 5 minutes at 4°C; one tube was lost in the centrifuge.
  4. Resuspended in 330 µl of 50 mM CaCl2 solution, 5 tubes w/ pellets; combined resuspension into 1x1.5mL tube.
  5. Centrifuged 5 minutes at 3000 RPM at 4°C.
  6. Poured off supernatant; resuspended pellet in 400 µl CaCl2.
  7. Placed on ice.

Transformation

Split 400 µl of suspended cells (see above) into 4 tubes of 100 µl.

Transformations:

  1. control: 1 µl pJExpress with amp-resistance and RFP.
  2. pGEM with 1 µl WillRS
  3. pNoTat with 1 µl WillRS (two tubes).
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