Melbourne/Lab Notebook gv 3

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Prepared for site dirrected mutagenesis

  • Produced NZY broth 100mL autoclaved (due to contamination in last batch).
  • Diluted DNA from last round
Tube conc of miniprep ng/ul total=(100ul) 1uL miniprep added to x uL milliQ
5A 141 522
6A 212 784
11A 221 817
10B 253 936

Site dirrected Mutagenesis Round #2

  • Applied the stratagene Site directed mutagenesis protocolto the following DNA and primer pairs, which when plated out on LB AMP plates producing the numbers of colonies shown. Several of these were picked and tubes marked with a character suffix as shown in table.

Changed PCR conditions on some primers that didn't work before to see if they would work. Specifically segment1 increased from 1 minute @ 95deg to 2minutes, segmet 2 increased from 50 sec @ 95 deg to 1 minute, and elongation time increased from 10 min @ 68 deg to 15 minutes.

Site dirrected Mutagenesis round #2
Mutation Number Template DNA (10ng) Sence Primer Antisence Primer #Colonies Picks named
21 5A GvpL-g351a GvpL-g351a-R 21A,21B
22 6A GvpL-g318a GvpL-g318a-R 3 22A,22B,22C
23 11A GvpQ-g183a GvpQ-g183a-R 26 23A,23B
24 10B GvpP-g441a GvpP-g441a-R 60 24A,24B
25 6A GvpL-g696a GvpL-g696a-R 1 25A,25B (new PCR conditions)
26 6A GvpL-g213a GvpL-g213a-R 26A,26B (new PCR conditions)
27 10B GvpQ-g150a GvpQ-g150a-R 27A,27B (new PCR conditions)
28 10B GvpQ-g150a GvpQ-g150a-R None
DNA concentrations in ng/uL
HistoryMutation tube\colony: A B C D E
pNL29T1-(Comp-g318a)-(GvpL-g351a)->21 183
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->22 176
pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->23 247
pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->24
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->25
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->26
pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->27
  • Diagnostic digests were performed using
    • A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2h30min. (25uL reaction)
    • B) 21.5uL of each p29T1 based colony in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U) at 37degC for 2h30min. (26uL reaction)
    • C) 21.5uL of each pNL26P3 based colony and in Buffer2 with 1uL EcoRI (20U) at 37degC for 2h30min. (25uL reaction)
  • Prepared two 20 lane 100mL agarose gel
  • Loaded 20uL of digest
  • Digest Pattern
  • Expected effects
PstI digest
HistoryMutation tube FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a)->21 5983 2516 483 <-
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->22 5983 2516 483 <-
pNL26P3-(GvpP-g441a)-(GvpQ-g183a)->23 4148 3170 2516 378 <- 105
pNL26P3-(GvpQ-g183a)-(GvpP-g441a)->24 4148 3170 2516 378 <- 105
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->25 5983 2894 <- 105
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->26 6088 2516 378 <-
pNL26P3-(GvpQ-g183a)-(GvpQ-g150a)->27 3928 3390 2516 378 <- 105
  • Results of PstI diagnostics:
    PstI digest
EcoRI & BamHI digests
EcoRI and BamHI digest
HistoryMutation tubeFragments:
pNL29T1-(Comp-g318a)-(GvpL-g351a)->21 6871 2112 <-
pNL29T1-(GvpL-g351a)-(GvpL-g318a)->22 6871 2112 <-
pNL29T1-(GvpL-g351a)-(GvpL-g696a)->25 6871 2112 <-
pNL29T1-(GvpL-g351a)-(GvpL-g213a)->26 6871 2112 <-
  • Results of EcoRI diagnostics:

Conclude 21A,22A,23A are all satisfactory.