Team:BYU Provo/Team Thermosensor/Week20

Week 20

August 29

Streaked four blue colonies from each: Lig56-2, 56-3, 57-1 to new plates. Incubating each colony at 30 celcius and 37 simultaneously.Expected results are that the positive controls will be blue at any temperature while the best thermosensors show drastic white-blue change between 30 and 37.

Low melt on RD 55-1, 56-1, 55-2 (40 micrometers/well) Now labeled gs55-1, gs56-1, gs55-2; put in TS freezer box

Was going to do ligation, but pIG 15 is all gone. Set up a plasmid prep using colonies from IGM 31 plate (Aug 26) 2(5ml LB amp) tubes labeled pIG 15 and a blank put in 37 degree shaker. Need to RD after plasmid prep and run on low melt so we can set up ligations.

Found “el problema” with our media recipe. Should be 15g agar, not 5g. So... Making plates correctly. 2L of media.

Bacto-tryptone: 20g

• Yeast: 10g
• NaCl: 10g
• Agar: 30g (!!!)
• Arabinose: 0.2g
• Amp: 2ml
• X-gal: 2ml

August 30

Blank not blank - must’ve been contaminated. So, just to be safe, set up new set of IGM 31 and blank and put in shaker at 37 again.

August 31

Did plasmid prep on 3 tubes with IGM31 following Qiagen kit protocol Nanodropped it like it’s hot and labeled tubes accordingly. All 3 tubes between 180 ng/ul and 321 ng/ul. My class starts in 10 minutes, so no time for RD. I’ll be back later.

RD - Vector digestion on pIG15 (IGM31)

• 14ul ddH20
• 5ul Buffer 2
• 0.5 ul BSA
• 30ul IGM31 - used IGM31 with nanodrop reading of 273 ng/ul
• 1.5 ul pSTI
• 1.5 ul HindIII

place in 37degree incubator at 3:55 pm

Repeated the above 3 more times with more IGM31

September 1

Low melt on RDpIG 15 1-3 and IGM 31 from previous page. Ligation of 55-1 and 55-2 (mutations) and 56-1 (Griffmed)

Mastermix (x6)

• 39ul H2O
• 9ul lox ligase buffer
• 6ul T4 DNA ligase
• 9 ul of Master Mix plus 3ul vector gs pIG15

Combine with:

• lig 55-1 and 3 ul gs55-1 (x2)
• lig 55-2 and 3 ul gs55-2 (x2)
• lig 56-1 and 3 ul gs56-2 (x2)

Transformation: plates T55-1, T55-2, and T56-1 Added 10 ul of ligation mix to DH5alpha tubes on ice Heat shocked 60 secs. Ice 3 mins Added 0.5 ml LB and incubated at 30 degrees for 30 minutes plated 100 ul and placed in 30 degree incubator overnight Set up additional ligations with varied ammounts of plasmid DNA and insert DNA

Lig 60-1

• 6.5ul H20
• 1.5ul 10x ligase buffer
• 1 ul DNA ligase
• 4.5 ul DNA insert (gs55-1)
• 1.5 ul plasmid DNA

Lig 60-2 Same as 60-1 except insert used—gs 55-2 Lig 60-3

• Same as lig60-1 except varied concentration of vector and insert.
• 3 ul insert DNA (gs55-1)
• 1 ul plasmid DNA

Lig 60-4

• Same as Lig 60-3, including concentration change, however gs55-2 used for insert DNA

Transformation of ligations 60-1, 60-2, 60-3, 60-4.

• Standard transformation protocol

September 3

Took 8 colonies from each T55-1, T55-2, and T56-1 (the two colonies containing our mutagenic thermosensor, and our Griffmed thermosensor)

Mutation T55-1

• Plated on T61-1 (30oC)
• Plated on T61-1 (37oC)

Mutation T55-2

• Plated on T61-3 (30oC)
• Plated on T61-4 (37oC)

T56-1 (GriffMed)

• Plated on T61-5 (30oC)
• Plated on T61-6 (37oC)