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Thursday, July 15 2010

Cast a 1% agarose gel; ran PCR product

• Lane 1 – Ladder (1 kb+)
• Lane 2 – 20 µl
• Lane 3 – 20 µl
• Lane 4 – 20 µl
• Lane 5 – 20 µl

With 5 µl loading dye to 50 µl reaction. Gel purified using Qiagen Kit. Eluted in 45 µl Buffer EB – two tubes.

• 4 colonies in four tubes of L. lactis with pPTPi into 5 mL MRS broth
• 1 tube has 5 µg/mL tet to test resistance
• 2 tubes of L. lactis without pPTPi in 5 mL MRS media.

13:42 incubation at 30°C.

Test tetracycline concentrations

Transformants from yesterday still grew in a lawn.

• Stock at 20 mg/mL
• 4 tubes LB of 10 µg/mL (2.5 µl stock), 12.5 µg/mL (3.125 µl stock), 15 µg/mL (3.75 µl stock), 17.5 µg/mL (4.375 µl stock).
• 1 tube of 150 µl BL21 split amongst the 4 tubes (~37 µl each tube)
• Incubate at 37°C overnight beginning at 14:15.

Followed Qiagen kit gel isolation/purification protocol of our PCR product (WillRS)

Ligation of WillRS and pGEM T Easy

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

15 µl total volume

Put at 4°C over the weekend – 36 hours.

Checked online sources – some other researchers say that it is okay to do a sticky end ligation overnight at room temperature. Followed Gary’s advice: leave overnight at 4°C (in fridge).

BBa_J06702

• Found 2 colonies on a transformed BL21 plate and 1 colony on an XL1B plate from yesterday’s transformation (realized that the competent cell solution was streaked onto the plates, rather than having the entire solution spread about the plates, so the number of cells on the plates was extremely to low to begin with—we are fortunate to have gotten these colonies).
• Recovered these colonies with inoculating loop and placed each into liquid media and into incubator so as to re-plate and create a glycerol stock of this part.

Transformations to test cell competence

Plasmids:

• Empty pNoTat (nanodropped to 83 ng/µl)
• Control pJExpress RFP (10 ng/µl)

Cells to test (all from the -80°C)

• BL21 from 6/24
• Xl1B from 6/24
• XLIB from 6/28

Added 100 pg of plasmid to each cell line tested (6X samples total). Transformation using Adrian 7/8/10 modified protocol. Plated on 6X Amp+ (100 µg/mL) plates.