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Revision as of 19:10, 19 July 2010

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Friday, June 25 2010

Motility

Took out James’ plates

RESULTS:

Strain	null plate	amp+IPTG	Chlor+arab
CONTROL	no growth	no growth	no growth
DFB 22	immotile growth	no growth	no growth
DFB 201	immotile growth	no growth	no growth
DFB 22 w/ pDB28	growth	no growth	no growth
DFB 201 w/ pDB101	immotile growth	no growth	motile growth
DFB 201 w/ pDB102	immotile growth	motile growth	no growth

Bold indicates not expected.

Also null bacteria streaked on BIOL 1220 amp+ plates still grew. This indicates that the amp plates from BIOL1220 are bad.

Also reviewed motility stabs – immotile strains DFB22 and DFB 201 seem immotile, but motile strains are not clear, which is okay as we still have 24 to 36 of incubation time to go.

It seems as though DFB 22 w/ pDB28 has consistent no growth, and so that strain is possibly nonviable (which is okay, as DFB is motB, not motAmotB, which is more interesting).

In general, it appears as though the stabs were more effective than the plates in inducing growth.

Miraculin

Lactobacillus acidophilus aliquoting

NCFM and 43121

43121 Each aliquot has 500 µl dH2O and 200 µl bacteria (+H2O)

NCFM “”

Plates:

• Null plates – 200 µl stock
• Chlor plate – 100 µl each

PCR of pWill1

• 2 µl forward primer
• 2 µl reverse primer
• 1 µl plasmid
• 25 µl master mix
• 20 µl dH2O

50 µl total volume

PCR program on thermocycler: “IGEM”

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Autoclaved LB + agar to make more chloramphenicol plates. Chloramphenicol at 15 µg/ml; made plates.

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Gave Gary pWill1 and pTG262

Ligation of ~850 bp amplified insert (denoted WillRS) into pGEM T Easy vector

pGEM is already linearized.

Used Nanodrop to find DNA concentration (insert) – 49.5 µg/µl?

   -Blank with master mix

We did not trust the Nanodrop readings because it began giving negative readings. So, we are unsure of the concentration and don’t want to do the ligation just yet.

Ran gel (1% Agarose) of PCR product

• Lane 1 – 1kb+ ladder (~20µl)
• Lane 2 – empty
• Lane 3 – Lily PCR (20µl)
• Lane 4 – Ethan PCR (20µl)

QIAGEN gel extraction

1. Cut out 2 bands (~850 bp, expected)

• • Weight: 0.19 g

1. Added 570 µl Buffer QG, followed through with protocol and eluted 50 µl buffer EB. (2 tubes, 100 µl total)

Ligation

• 1 µl 10X T4 DNA ligase buffer
• 1 µl pGEM T Easy (~3kb)
• 3 µl insert (~850 bp)
• 4.5 µl dH2O
• 0.5 T4 DNA Ligase

10 µl total volume

RT for 0.5 hr Then 65°C to denature ligase

Product put into WRD freezer box.

Control ligations:

1. No insert; dH2O instead
2. No vector; dH2O instead