Melbourne/Lab Notebook gv 4

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Prepared for site dirrected mutagenesis

  • Diluted DNA from last round to give 10ng/37ul
Tube conc of miniprep ng/ul total=(100ul) x uL miniprep added to y mL milliQ
21A 192 2ul in 1.42ml
22A 176 2ul in 1.3ml
23A 247 1ul in 0.91ml

Site dirrected Mutagenesis Round #3

  • 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.
  • PCR conditions:
    • seg 1 95 degC 2min x 1
    • seg 2 x 18
      • 95 deg 1min
      • 60 deg 50 sec
      • 68 deg 15min
    • seg 3 68 deg 7min x 1
    • seg 4 4 deg indefinately
Site dirrected Mutagenesis round #3
Mutation Number Template DNA (10ng) Sence Primer Antisence Primer #Colonies Picks named
31 21A GvpL-g696a GvpL-g696a-R lots 31A,31B
32 22A GvpL-g213a GvpL-g213a-R lots 32A,32B
33 23A GvpQ-g150a GvpQ-g150a-R lots 33A,33B
DNA concentrations in ng/uL
HistoryMutation tube\colony: A B
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)->31 117 133
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)->32 107 96
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)->33 114 152
  • Diagnostic digests were performed using
    • A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 1h30min. (25uL reaction)
  • Prepared two 8 lane 50mL agarose gel
  • Loaded 20uL of digest
  • Digest Pattern
  • Expected effects
PstI digest
HistoryMutation tube FRAGMENTS
pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)->31 5983 2999 <- -
pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)->32 6466 2516 <- -
pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)->33 7318 <- 2516 378 - 105
  • Results of PstI diagnostics

Conclude 32B and 33A are good, and 31A & B are disturbingly different but similar so we should take both of them forward. Poor Jell though.