Team:University of Lethbridge/Notebook/GeneralLabAugst

From 2008.igem.org

                         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

August 02, 2008

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

August 03, 2008

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

August 5, 2008

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

August 6, 2008

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.

August 7, 2008

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