Team:University of Lethbridge/Notebook/Project2August

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Nathan Puhl, Roxanne
Riboswitch 20 uL PCR. Set up 4 reactions (25 uL for each total volume). 1 uL or 1/100 pTopp and 1 uL or H1/100 PCR from July 28, 2008.

PCR conditions: A. Initial denaturation: 98 C (3 min) B. -Denaturation: 98 C (10 sec) - Annealing: 55 C (30 sec) -Extension: 72 C (15 sec) -30 cycles C. Final extension: 72 C (7 min)

Nathan Puhl, Roxanne
Ran riboswitch (Aug. 1, 2008) on 3% agarose gel. Results are in the hard copy lab notebook. Looks like 76 bp band will extract.

Nathan Puhl, Roxanne
Riboswitch:

Set up PCR using purified riboswitch from Aug. 5, 2008 with platinum Taq (50 uL reaction). Made Master Mix for three reactions. Master Mix: -10x Buffer (no Mg2+): 15 uL -10 mM dNTPs: 3 uL -50 mM Mg2+: 4.5 uL -10uM RF: 3 uL -10uM RR: 3 uL -Plat. poly: 0.6 iL -H20: 120.9 uL -template: 1 uL

Cycle conditions: A. Initial denaturation: 94 C (2 min) B. -Denaturation: 94 C (30 sec) - Annealing: 55 C (30 sec) -Extension: 72 C (30 sec) -30 cycles C. Final extension: 72 C (7 min)

Roxanne
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.

Nathan Puhl, Roxanne, Munima
-Restriction Digested the purified riboswitch and pSB1A7 with XbaI and SpeI, let it run for 4 hours

Nathan Puhl, Roxanne
-Ran all of the restricted pSB1A7 plasmid through a 1% agarose gel at 100V for 25 minutes. -Ran a gel extraction on the pSB1A7 cut plasmid, and ran a PCR clean-up reaction on the digested RS1 and RS2 amplicons. -Ran 1 uL of each on a 1% to quantify the amount of DNA present. -Ligated RS1 + pSB1A7, and RS2 + pSB1A7, using T4 DNA Ligase.

-1 uL of RS1/RS2 -4 uL of pSB1A7 -1 uL of 10x T4 DNA Ligase Buffer -0.33 uL of T4 DNA Ligase -3.67 uL of water

allowed the reaction to go overnight

Nathan Puhl
-Transformed DH5a cells with the pSB1A7 + RS1, and pSB1A7 + RS2 plasmids.

-Plated on semi-solid agar plates containing 100 ug/mL of ampicillin.

Christa, Nathan Puhl, Munima
-Nathan checked the plates for growth. Colonies are present.

-Ran a colony PCR of the pSB1A7 + RS1, and pSB1A7 + RS2 recombinant cells transformed by Nathan and Roxanne on August 17.

-Inoculated the cells into tubes of liquid media + 100 ug/ml of ampicillin. Roxanne will remove the PCR tube from the thermocycler in the morning.

Roxanne
-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.



-Plasmid Prepped and made glycerol stocks from the RS1-1 and RS2-1 tubes of cells incubated in LB media + 100 ug/mL ampicillin.

Ran the pRS1 and pRS2 plasmids on a 1% Agarose Gel at 100 V for 30 minutes.



Nathan Puhl, Roxanne
-Screened the pSB1A7 + RS1, and pSB1A7 + RS2 by PCR using the VF2 and RS1/RS2 Reverse Primers determine whether the plasmids obtained from the recombinant cells contain the riboswitch, and if so, if it inserted in the correct orientation.

Roxanne
-Ran the PCR products on a 1% Agarose Gel at 100 V for 33 minutes. The gel was empty with the exception of primer dimers.

Nathan Puhl, Roxanne
-went over the SELEX protocol with HJ to determine the primers we will need to do this, and how exactly we plan on perfoming the evolution.

-setup a restriction digest for pSB1A7 using XbaI and SpeI, ran overnight.

Nathan Puhl, Roxanne
-Digested pSB1A7 with Antarctic Phosphatase

-9 uL of cut pSB1A7 -1.5 uL of 10x Antarctic Phosphatase Buffer -1 uL of Antarctic Phosphatase Enzyme -3.5 uL of water Allowed the Reaction to take place for 30 minutes to remove the 5` Phosphates from the pSB1A7 plasmid to prevent religation.

-Ran the remainder of the pSB1A7 plasmid from August 22nd on 1 1% Agarose Gel at 100 v for 27 minutes.

-Gel Extracted the plasmid DNA.

-Purified the Phosphatase reaction to isolate the pSB1A7 DNA.

-Ran a 1% gel to quantify the amount of plasmid DNA present.

-Ligated RS1 and RS2 into the dephosphorylated pSB1A7 using T4 DNA Ligase.

-1 uL of RS1 or RS2 -4 uL of dephosphorylated pSB1A7 -1 uL of 10X T4 DNA Ligase Buffer -0.33 uL T4 DNA Ligase Enzyme -3.67 uL water Reaction was allowed to go overnight

Nathan Puhl
-Transformed DH5a cells with the RS1+pSB1A7 or RS2+pSB1A7 plasmid on semi-solid agar plates containing 100 ug/mLof ampicillin.

Roxanne, Nathan Puhl, Munima, Sebastian, Andrew
-Performed the ligation of the riboswitch into pGEM T-easy, transformed and plated.

Nathan Puhl, Roxanne, Andrew
-Performed a colony PCR on representative colonies containing the pGEM T-easy plasmid to screen for the presence of the riboswitch.

Same as previous protocol, However, Annealing Temperature is set to 65.0 C

-Ran a gel of the PCR products on 3% Agarose @ 100V for 27 minutes.

Roxanne
-Reran the gel of the colony PCR, determined that it did not work.

-Performed a second colony PCR on 3 white colonies, 1 blue colony and 1 blue/white colony from the pGEM T-easy + RS1/RS2 recombinant cells.

Same as Previous Protocol, annealing temperature is 50.0C

-Ran a 2% Agarose Gel of the PCR Products at 100V for 27 minutes.

-One of the White Colonies and the Blue/White Colony in each case amplified.