Team:Newcastle/Labwork/25 September 2009

=Formal Lab Session - 25th September 2009=

Introduction

 * To obtain pspac part from pMutin4, we digested psc fragment with HindIII and BamHI
 * We need Mini prep kinA transformation and check the result.

Digestion

 * Digest psc clean-up DNA

dd H2O                    7ul 10X fast digest buffer    7ul Fast HindIII              3ul Fast BamHI                3ul psc clean-up DNA         50ul --                           70ul


 * Incubate 1 hour at 37 degree

Gel Extraction

 * After the digestion of psc fragment we run the digested DNA on big well gel and cut the right band and purified the DNA by gel extraction kit.

Mini prep

 * Followed the Phil's protocal.
 * Since we cultured 24 colonies yesterday, we performed Mini Prep for all those 24 strains.

Test the Mini Prep result
dd H2O                    7ul 10X fast digest buffer    2ul Fast EcoRI              0.5ul Fast NheI               0.5ul Mini Prep DNA            10ul --                           20ul
 * Digested the Mini Prep DNA with EcoRI and NheI
 * 37C incubated for 1 hour
 * Add 2ul stop buffer and run the sample on agarose gel.

Ligation
ddH2O                            10ul ligation buffer                   4ul psc fragments                    10ul pMutin4 backbone (24.9ng/ul)      5ul T4 ligase                         1ul --                                   30ul
 * Ligate psc fragment with pMutin4 backbone

conclusion

 * After the digestion of kinA transformation Mini Prep result, it seems No.4,5,6,10 could be the right clone. So, we prepare Midi culture for all 4 strains.



Futher plan

 * Use HindIII enzyme digest Mini prep result of those 4 strains to double check the result.