Formal Lab Session - 1st October 2009

Restriction digest tests

Our previous transformaitons did not work well. We we tested if we had the right pmutin4 and cotC biobricks.

We run them on the gel.

pmutin4:

4ul DNA
5ul Water
1ul loading buffer

CotC:

3ul of DNA
6ul DNA
1ul loading buffer

The bands were right. However somce BamHI and HindIII sites on pmutin4 are too close, the backbone may not have been cut properly.

Preparation of competent cells

We prepared fresh DH5 alpha cells by plating them into two new LB plates

We also prepared a set of new E.coli DH5 alpha competent cells. we measured the ODs of E. coli cells during transformation. We waited 2 hours before measrung the ODs and we get the absorbance result as 0.146. To measure the ODs

Set the device to 600nM
Place a tube with LB only and take a reference measurement
Place the culture and press the green box.

We then tested our competent cells for transformation. We used 40ul of cells + 3ul DNA for the tests. We used pmutin4 and pSB1AT3 for the tests

Ligations

We prepared ligations for Cot + pmutin4. Used two different versions og pmutin4 and prepared the ligations below

cotc + pmutin4 cut1
cotC + pmutin4 cut2
psc + pmutin4 cut 2

We also prepared controls as below

backbone + ligase + no insert
backbone + no liase + no insert

Chassis team

Introduction

Digest pMK-RQ:kinA and pGFP-rrnB:kinA with same enzyme and run DNA on agarose to check whether our Midi prep is the right one.

Experiment procedure

Digest pMK-RQ:kinA and pGFP-rrnB:kinA

EcoRI and NheI were used for digestion.

 dd H2O                     7ul
 10X fast digest buffer     2ul
 Fast EcoRI               0.5ul
 Fast NheI                0.5ul
 pMK-RQ:kinA               10ul
 ------------------------------
                           20ul

 dd H2O                     7ul
 10X fast digest buffer     2ul
 Fast EcoRI               0.5ul
 Fast NheI                0.5ul
 pGFP-rrnB:kinA            10ul
 ------------------------------
                           20ul