Team:Newcastle/Labwork/1 October 2009

=Formal Lab Session - 1st October 2009=

Restriction digest tests
Our previous transformations did not work well. 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 some 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 of pMUTIN4 and prepared the ligations below
 * 1) cotC + pMUTIN4 cut1
 * 2) cotC + pPMUTIN4 cut2
 * 3) psc + pMUTIN4 cut 2

We also prepared controls as below
 * 1) backbone + ligase + no insert
 * 2) backbone + no liase + no insert

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.

Digest pMK-RQ:kinA and pGFP-rrnB:kinA
dd H2O                    7ul 10X fast digest buffer    2ul Fast EcoRI              0.5ul Fast NheI               0.5ul pMK-RQ:kinA              10ul --                           20ul
 * EcoRI and NheI were used for digestion.

dd H2O                    7ul 10X fast digest buffer    2ul Fast EcoRI              0.5ul Fast NheI               0.5ul pGFP-rrnB:kinA           10ul --                           20ul
 * 37C 1 hour.
 * Run the sample on 0.8% agarose gel.

Conclusion
lane 1: lamda ladder lane 2: 1kb ladder lane 3: pMK-RQ:kinA lane 4: pGFP-rrnB:kinA (our NO.6 in transformation)
 * The gel picture showed that our kinA clone was not right.