Team:University of Lethbridge/Notebook/Project2August

From 2008.igem.org

(Difference between revisions)
m
m
Line 43: Line 43:
====Roxanne====
====Roxanne====
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.
-Ran the PCR Product on a 3% gel using only 1uL of DNA from the riboswitch.
 +
 +
===August 16, 2008===
 +
====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
 +
 +
 +
===August 17, 2008===
 +
====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.
 +
 +
 +
===August 18===
 +
====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.
 +
 +
 +
===August 19, 2008===
 +
====Roxanne====
 +
-Ran the PCR Product on a 2% Agarose Gel at 100 V for 33 minutes.
 +
 +
[[Image:RS1 RS2 gel.jpg| 350 px]]
 +
 +
-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.
 +
 +
[[Image:pRS1 pRS2 gel.jpg| 150 px]]

Revision as of 23:16, 3 September 2008

Back to The University of Lethbridge Main Notebook


Contents

August 1, 2008

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)

August 2, 2008

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.

August 7, 2008

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.

August 16, 2008

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


August 17, 2008

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.


August 18

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.


August 19, 2008

Roxanne

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

RS1 RS2 gel.jpg

-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.

PRS1 pRS2 gel.jpg