SBB10AssayTeam4-Notes

Carolyn Kwok, Jing Luo, Dorothy Tulanont

Goals

1. Determine whether the N15 protelomerase parts are functional or not on the Tos site assuming the co-transformation is stable.
2a. If the plasmids don't stably co-transform, determine whether cells containing N15 protelomerase enrich for plasmids lacking tos.
2b. Assuming the N15 protelomerase parts are functional, determine whether the linearized "doggy bone" re-circularized in the absence of N15 protelomerase.

General Procedure

Part 1
Experiment:
1. Cotransform cells with plasmid containing the tos site and N15 protelomerase expressing plasmid.
2. Heat-shock
3. Plate with resistance plate
4. Miniprep cells
5. Digest the resulting plasmid with a restriction enzyme.
6. Analytical Mapping
Controls:
1. Transform the cells with only the Tos site plasmid
2. Run the undigested cotransformed DNA.

Part 2a
Experiment:
1. Transform cells with N15 plasmids
2. Plate on antibiotic for N15
3. Pick colonies
4. Mix Tos plasmid and RFP plasmid at a 100:1 ratio
5. Transform mixture into cells
6. Plate on antibiotics for Tos, RFP, and N15 (plate 1)
7. Count ratio of white colonies to red colonies
Control:
1. Mix Tos plasmid and RFP plasmid at a 100:1 ration, transform into cells without N15 and plate (plate 2)
Calculation:
Enrichment = % of red in plate 1/% of red in plate 2

Part 2b
1. Prepare "doggy bone" plasmid and the non-linearized plasmid.
2. Transform "doggy bone" plasmid and the non-linearized plasmid into cells with N15 and cells without N15 (the wild type cells).
3. Count colonies on all 4 plates.

4/12/10

Perform quikchange procedure on tos plasmid to correct the point mutation.

4/19/10

Notes from last experiment: Our bacteria failed to grow. We have to change the Quikchange protocol.
Dorothy: Digest the vector with pBad and N15 plasmid with BglII and XbaI. Next time --> gel purification of these parts and we can proceed on to ligation.

Digestion of vector (pWCD 0011)
3 uL of water
1 uL NEB2+ATP buffer
5 uL of vector
0.5uL of  BglII
0.5uL of XbaI

Digestion of insert (N15) 
3 uL of water
1 uL of NEB2+ATP buffer
5 uL of insert
0.5uL of  BglII
0.5uL of XbaI

Jing: Starts the Quikchange (running it in the PCR machine). Control will be transforming bacteria without DpnI digestion.

The oligos are lost. Dorothy will come into the lab to start the PCR for quikchange on Tu 4/20 around 6PM.

4/20/10

Dorothy finished quikchange successfully.

4/21/10

We gel purified tos. For N15 (sbb01), we ligate, transform and plate.

4/22/10

Dorothy picked three tos colonies and culture them.

4/23/10

Carolyn miniprepped the three tos bacteria. Dorothy and Jing ran an analytical gel of tos. We sent the first clone to sequence for verification that the incorrect nucleotide has changed.