From 2007.igem.org
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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
- 95 deg 1min
- 60 deg 50 sec
- 68 deg 15min x18
- seg 3 68 deg 7min
- 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
| History | Mutation 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
| History | Mutation 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.
PstI digest
