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Tuesday, July 13 2010

Double digested Gary’s WillRS ligation, iGEM WillRS ligation, and pNoTat

Reagents used for 20 µl double digest:

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

20 µl total volume

1 hour in 37°C incubator from 11:00 AM

Inspection of results from last night’s transformation

All kanamycin plates are already filled with a lawn of bacteria, while ampicillin plates are not. Tentatively thinking that the LB/Agar that was prepared was contaminated with a kanamycin-resistant strain.

Transformation of pPTPi

• 4 tubes BL21 (150 µl)
• 2 tubes with 2.5 µl pPTPi
• 2 tubes with 1.0 µl RFP control

13:53 – incubate 30 minutes.

Heat shock 1 minute

Incubate 2 minutes on ice

Add 100 µl LB and plate on 10 µg/mL plates after letting cells recuperate for 30 minutes at 37°C

50 of 10 µg/mL plates: 720 µl of 12.5 mg/mL stock into 900 mL of LB

E.coli need to be grown on 10 µgmL tet! (L. lactis 5 µg/mL tet)

Ran a 1% gel of digest from earlier this morning:

• Lane 1: Ladder (1 kb+)
• Lane 2: pNoTat digest Expect 3 kb
• Lane 3: Gary pGEM digest 3 kb and 800 fragment
• Lane 4: iGEM pGEM digest 3 kb and 800 fragment

Run at 100V. Started at 2:45 PM.

Ran it for a bit too long -- see gel:

1. kb  interpretation: one digest worked.

Interpretation: After looking at the ladder (1kb+) we determined we couldn’t see an ~800 bp fragment although pGEM and pNoTat were ~3kb, as expected. This means that our blue-white selection white colonies actually did not have the insert.

To confirm/investigate this possibility, we decided to take Will primers and run a PCR to see if we would get any product (pWill1).

PCR of supposedly ligated pGEM (both iGEM-pGEM and Gary-pGEM) using Will’s primers (labeled RER and REF)

(2X – one for iGEM pGEM, another for Gary pGEM)

• 2 µl fwd primer
• 2 µl rev primer
• 1 µl pGEM (plasmid)
• 25 µl PCR master mix
• 20 µl dH2O

50 µl total volume

PCR program on thermocycler: “IGEM” Start at 7:00 PM.

• Took ~3kb band from gel, pNoTat lane
• Gel extraction protocol on 122 mg of gel
• Stored product as “digested pNoTat, 7/13”