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| - | <div align="center">Week 11: Monday 27th September - Sunday 3rd October</div>
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| - | ==Monday==
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| - | ===114. Expt: Continuation of cultures for Mexico (Peter)===
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| - | Sent to Mexico using Interparcel/DHL.
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| | | | |
| - | Tracking number 903532038716
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| - |
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| - | Paid for by Peter: £26.50
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| - |
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| - | ===115. Expt: Biobrick assembly of fluorescent proteins (YFP, CFP, RFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily and Bill)===
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| - | *Miniprepped overnight cultures of YFP, CFP, RFP with rbs's and PP+pBAD+pSB1C3
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| - | *Nanodrop readings:
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |Nanodrop reading (ng/µl)
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| - | |-
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| - | |Cm+PP+pBAD+pSB1C3
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| - | |48.5
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| - | |-
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| - | |YFP+rbs
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| - | |74.8
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| - | |-
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| - | |RFP+rbs
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| - | |32.9
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| - | |-
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| - | |CFP+rbs
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| - | |46.7
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| - | |}
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| - |
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| - | pSB1C3 from freezer was used at 12.9ng/µl
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| - |
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| - | *Restrict using protocol on p88, using following quantities:
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |Cm+PP+pSB1C3
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| - | |YFP (for PP)
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| - | |YFP (for pBAD)
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| - | |RFP (for PP)
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| - | |RFP (for pBAD)
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| - | |CFP (for PP)
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| - | |CFP (for pBAD)
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| - | |pSB1C3
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| - | |-
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| - | |Nuclease-free H20
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| - | |1
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| - | |6
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| - | |6
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| - | |1
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| - | |1
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| - | |6
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| - | |6
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| - | |1
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| - | |-
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| - | |10x FD Buffer
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |-
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| - | |Plasmid DNA
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| - | |15
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| - | |10
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| - | |10
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| - | |15
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| - | |15
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| - | |10
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| - | |10
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| - | |16
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| - | |-
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| - | |EcoRI
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| - | |0
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| - | |0
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| - | |0
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| - | |0
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| - | |0
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| - | |0
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| - | |0
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| - | |1
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| - | |-
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| - | |SpeI
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| - | |0
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| - | |1
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| - | |0
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| - | |1
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| - | |0
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| - | |1
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| - | |0
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| - | |0
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| - | |-
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| - | |XbaI
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| - | |1
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| - | |0
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| - | |1
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| - | |0
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| - | |1
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| - | |0
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| - | |1
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| - | |0
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| - | |-
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| - | |PstI
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |-
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| - | |
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| - | |*
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| - | |*
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| - | |
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| - | |*
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| - | |
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| - | |*
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| - | |
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| - | |
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| - | |}
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| - |
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| - | *We ran this gel on an E-gel and realised that those lanes with '*' had been cut with the wrong enzymes - PP+pSB1C3 should have been cut with S+P, the RFPs should have been cut with X+P.
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| - | *We carried on with those that had worked with just pBAD.
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| - |
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| - | See continuation below.
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| - |
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| - | ===116. Expt: Plate reader readings for Arabinose induction for LCLuc+PPLuc under pBAD===
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| - |
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| - | Arabinose concentration varying from 0µM to 10mM
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| - |
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| - | *1. 0µM: 0Ara/30µl of H20
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| - | *2. 1µM: 1µl of Ara@100µM/29µl of H20
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| - | *3. 3µM: 3µl of Ara@100µM/27µl of H20
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| - | *4. 10µM: 10µl of Ara@100µM/20µl of H20
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| - | *5. 30µM: 30µl of Ara@100µM/no H20
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| - | *6. 100µM: 1µl of Ara@10mM/29µl of H20
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| - | *7. 300µM: 3µl of Ara@10mM/27µl of H20
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| - | *8. 1mM: 10µl of Ara@10mM/20µl of H20
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| - | *9. 3mM: 30µl of Ara@10mM/no H20
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| - | *10. 10mM: 1µl of Ara@1M/29µl of H20
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| - | *11. No luciferin, 100µM Ara => 1µl of 10mM, 30.5 of H20
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| - |
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| - | D-luciferin at 100µM -> 15µl.
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| - |
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| - | Total volume 100µl per well. 60.5µl of overnight culture
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| - |
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| - | Plate layout:
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |1-3
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| - | |4-6
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| - | |7-9
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| - | |10-12
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| - | |-
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| - | |A
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| - | |PP(1)
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| - | |PP(2)
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| - | |PP(3)
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| - | |PP(4)
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| - | |-
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| - | |B
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| - | |PP(5)
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| - | |PP(6)
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| - | |PP(7)
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| - | |PP(8)
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| - | |-
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| - | |C
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| - | |PP(9)
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| - | |PP(10)
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| - | |PP(11)
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| - | |LC(5)
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| - | |-
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| - | |D
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| - | |
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| - | |
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| - | |
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| - | |
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| - | |-
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| - | |E
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| - | |
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| - | |
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| - | |
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| - | |
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| - | |-
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| - | |F
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| - | |LC(1)
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| - | |LC(6)
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| - | |LC(3)
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| - | |LC(4)
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| - | |-
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| - | |G
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| - | |
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| - | |
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| - | |LC(7)
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| - | |LC(8)
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| - | |-
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| - | |H
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| - | |LC(9)
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| - | |LC(10)
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| - | |LC(11)
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| - | |Blanks
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| - | |}
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| - |
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| - | ===117. Expt: Repeat of Ben's experiment of altering the other 4 sites of LC luciferase using directed mutagenesis (Will)===
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| - | PCR mixes were:
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| - | *0.25µl forward primer
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| - | *0.25µl reverse primer
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| - | *2µl template
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| - | *22.5µl PCR water
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| - | *25µl 2x Phusion Mastermix
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| - |
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| - | *Template DNA was taken from colony (100µl PCR water + 1 colony)
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| - | *Primers were taken directly from tubes (undiluted)
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| - |
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| - | ====PCR Protocol====
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |110°C
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| - | |Heated lid
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| - | |-
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| - | |1m30
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| - | |98°C
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| - | |Denaturation
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| - | |-
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| - | |Cycle 30 times
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| - | |
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| - | |
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| - | |-
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| - | |30s
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| - | |98°C
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| - | |Denaturation
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| - | |-
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| - | |2m
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| - | |72°C
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| - | |Annealing & Elongation
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| - | |-
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| - | |End cycle
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| - | |
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| - | |
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| - | |-
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| - | |7m30
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| - | |72°C
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| - | |Final elongation
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| - | |-
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| - | |
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| - | |10°C
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| - | |Final hold
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| - | |}
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| - |
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| - | Did not work - abandon experiment
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| - |
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| - | ===118. Continuation of Biobrick assembly of fluorescent proteins (YFP, RFP, CFP) into PP+pBAD+pSB1C3 and pBAD+pSB1C3 (Emily)===
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| - | *Lengths of proteins cut with X+P were right
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| - | *Gel extraction was performed using Qiagen protocol
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| - |
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| - | ====Ligation====
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| - | Followed protocol on p91 using following quantities:
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |RFP
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| - | |CFP
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| - | |YFP
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| - | |-
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| - | |5x Rapid Ligation Buffer
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| - | |4
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| - | |4
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| - | |4
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| - | |-
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| - | |T4 DNA Ligase
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| - | |1
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| - | |1
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| - | |1
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| - | |-
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| - | |pSB1C3
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| - | |5.7
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| - | |5.4
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| - | |6.2
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| - | |-
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| - | |DNA
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| - | |8.1
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| - | |8.5
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| - | |7.5
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| - | |-
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| - | |pBAD (34.5ng/µl)
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| - | |1.2
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| - | |1.1
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| - | |1.3
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| - | |}
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| - |
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| - | *Cells were transformed on 28/9/10 overnight after leaving them to air under fume hood.
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| - |
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| - | ==Tuesday==
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| - | *Restriction again, repeating from 27/9/10 using the correct restriction enzymes:
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |Cm+PP+pSB1C3
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| - | |YFP (for PP)
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| - | |RFP (for PP)
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| - | |CFP (for PP)
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| - | |-
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| - | |Nuclease-free H20
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| - | |1
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| - | |6
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| - | |6
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| - | |6
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| - | |-
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| - | |10x FD Buffer
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| - | |2
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| - | |2
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| - | |2
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| - | |2
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| - | |-
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| - | |Plasmid DNA
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| - | |15
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| - | |10
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| - | |10
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| - | |10
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| - | |-
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| - | |SpeI
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| - | |1
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| - | |0
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| - | |0
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| - | |0
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| - | |-
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| - | |XbaI
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| - | |0
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| - | |1
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| - | |1
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| - | |1
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| - | |-
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| - | |PstI
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| - | |1
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| - | |1
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| - | |1
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| - | |1
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| - | |}
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| - |
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| - | *All showed correct bands on gel
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| - | *Gel extraction was performed
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| - | *Ligation was performed:
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| - |
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |YFP (4.8ng/µl)
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| - | |RFP (12.1ng/µl)
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| - | |CFP (9.5ng/µl)
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| - | |-
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| - | |5x Rapid Ligation Buffer
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| - | |4
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| - | |4
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| - | |4
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| - | |-
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| - | |T4 DNA Ligase
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| - | |1
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| - | |1
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| - | |1
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| - | |-
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| - | |DNA
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| - | |4.7
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| - | |2.3
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| - | |2.8
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| - | |-
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| - | |PP+pSB1C3
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| - | |10.3
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| - | |12.7
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| - | |12.2
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| - | |}
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| - |
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| - | *Transformation into TOP10cc from freezer on Cm+Ara plates
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| - | *Also transformed plambda+rbs from registry onto Cm plate. Should have been on Amp plate so did not grow.
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| - |
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| - | ====Results====
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| - | *After 1 day - little colonies on all plates
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| - | *After 2 days - looks like lots of cross-contamination. Only some colonies glow. Streaked out interesting colonies + grew up overnight culture
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| - |
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| - | ==Wednesday==
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| - | ===119. Expt: Plate reader of:===
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| - | *bacteria containing PP luciferase under pBAD at varying arabinose concentrations, with & without LRE as part of the operon
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| - | *bacteria containing:
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| - | **LC wt
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| - | **LC 239
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| - | **LC 326
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| - | **LC 433
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| - | **LC 452
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| - | **EPIC
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| - | *Arabinose concentrations were set up as on p109
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| - | *D-luciferin at 10mM, 1µl were added
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| - | *Cells were taken from liquid culture apart from ... which was taken from solid culture into LB
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| - |
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| - | *Layout (PP LRE = luciferase with LRE, PP = luciferase without LRE):
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| - | {|class="wikitable"
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| - | |-
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| - | |
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| - | |1-3
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| - | |4-6
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| - | |7-9
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| - | |10-12
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| - | |-
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| - | |A
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| - | |PP LRE (1)
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| - | |PP LRE (2)
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| - | |PP LRE (3)
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| - | |PP LRE (4)
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| - | |-
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| - | |B
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| - | |PP LRE (5)
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| - | |PP LRE (6)
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| - | |PP LRE (7)
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| - | |PP LRE (8)
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| - | |-
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| - | |C
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| - | |PP LRE (9)
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| - | |PP LRE (10)
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| - | |PP LRE (11)
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| - | |
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| - | |-
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| - | |D
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| - | |PP (1)
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| - | |PP (2)
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| - | |PP (3)
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| - | |PP (4)
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| - | |-
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| - | |E
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| - | |PP (5)
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| - | |PP (6)
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| - | |PP (7)
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| - | |PP (8)
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| - | |-
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| - | |F
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| - | |PP (9)
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| - | |PP (10)
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| - | |PP (11)
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| - | |
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| - | |-
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| - | |G
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| - | |LC wt
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| - | |LC 239
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| - | |LC 326
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| - | |LC 433
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| - | |-
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| - | |H
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| - | |LC 452
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| - | |EPIC
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| - | |
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| - | |LB+water
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| - | |}
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"
