Team:University of Lethbridge/Notebook/GeneralLabAugst

Nathan Puhl, Nathan Phillips
Objective: Amplify more of the riboswitch using amplicons from the previous PCR MasterMix: 1x       4x 5x buffer    5 uL      20 10 mM dNTP   0.5 uL    2 RF primer    2.5 uL    10 RR primer    2.5 uL    10 Phusion      0.25 uL   1 ddH2O        15.25 uL  61 Template     1 uL of pTOPP, 1x, 1/10x riboswitch amplicon

Cycle Conditions: 98 C - 3 min 30x: 98 C - 10 sec 55 C - 30 sec 72 C - 15 sec 72 C - 7 min

Nathan Puhl
Ran gel of PCR products from Aug. 01 with pTOPP template in lane 2, 1x, 1/10x amplicon template in lane 3 and 4



Nathan Phillips
Tranformation of pUC19: 1. Thaw E.coli DH5a cells on ice 2. Add pUC19 DNA, pipette gently to mix (1μl of plasmid) 3. Let sit for 30 minutes on ice 4. Incubate cells for 30 seconds at 42oC 5. Incubate cells on ice for 2 min 6. Add 1 mL SOC at room temp 7. Incubate for 1 hour at 37oC on shaker 8. Spread 100-300 μl onto a plate made with appropriate antibiotic 9. Grow overnight at 37 °C

Nathan Puhl, Alix, Roxanne and highschool students
Objective: Isolate 76bp band from non-specific amplified bands of riboswitch PCR

Using Qiagen MinElute Kit extracted two very, very similiarly sized bands after running on a 3.5% agarose gel.



Objective: Cut out LacI gene and open DT vector for construction

Restriction Digest of BBa_C0012 (LacI) and BBa_C0012 (Double Terminator)

LacI - EcoR I + Spe I

DT - EcoR I + Xba I

Overnight incubation at 37 C followed by 65 C for 15 min (enzyme deactivation)

Reaction Mixture: -5 uL template -5 uL NEB 2 buffer -0.5 uL BSA -1 uL restriction enzyme #1 -1 uL restriction enzyme #2 -37.5 uL ddH2O

Nathan Puhl, Roxanne, Alix
Objective: Gel extract digested biobricks to clean for ligation reaction

Qiagen MinElute Kit was used to gel purify the digested LacI and DT biobricks.

Nathan Puhl, Roxanne, Peter
Objective: Amplify riboswitch with A overhangs for TA cloning

Set up PCR using purified riboswitch amplicon from Aug. 05 (1 uL of template)

MasterMix: 1x       3x 10x buffer   5 uL      15 10 mM dNTP   1 uL      3 50 mM Mg2+   1.5       4.5 RF primer    1 uL      3 RR primer    1 uL      3 Platinum taq 0.2 uL    0.6 ddH2O        39.3 uL   117.9 Template     1 uL of riboswitch amplicon

Cycle Conditions: 94 C - 2 min 30x: 94 C - 30 sec 53 C - 30 sec 72 C - 30 sec