From 2007.igem.org
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Prepared for site dirrected mutagenesis
- Diluted DNA from last round, creating 10ng/ul sample and then adding 1uL of this to 36uL milliQ water to produce required 10ng/37uL template for PCR.
| Tube | conc of miniprep ng/ul total=(100ul) | 1uL miniprep added to x uL milliQ -> 10ng/uL
|
| 31A | 117 | 10.7
|
| 31B | 133 | 12.3
|
| 32B | 96 | 8.6
|
| 33A | 114 | 10.4
|
Site dirrected Mutagenesis Round #4
- 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.
Site directed Mutagenesis round #3
| Mutation Number | Template DNA (10ng) | Sence Primer | Antisence Primer | #Colonies | Picks named
|
| 41 | 31A | GvpL-g213a | GvpL-g213a-R | lots | 41A,41B,41C
|
| 42 | 42A | GvpL-g213a | GvpL-g213a-R | lots | 42A,42B
|
| 43 | 32B | GvpL-g696a | GvpL-g696a-R | lots | 43A,43B
|
| 44 | 33A | GvpA-t57c | GvpA-t57c-R | lots | 44A,44B
|
DNA concentrations in ng/uL
| History | Mutation tube\colony: | A | B | C
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 41 | 111 | 115 | 129
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 42 | 133 | 129
|
| pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)-> | 43 | 107 | 75
|
| pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)-> | 44 | 221 | 204
|
- Diagnostic digests were performed using
- A) 21.5uL of each in Buffer3 with 1uL PstI (20U) at 37degC for 2hrs. (25uL reaction)
- B) 5uL of each in Buffer2 with 1uL EcoRI (20U) and 1uL BamHI (20U) with 16.5uL of milliQ at 37degC for 2h. (26uL reaction) except pNL26 based 44A,B which were digested with EcoRI only.
- C) 10 uL of each in Buffer2 with 2uL HindIII (20U) with 11.5 uL milliQ at 37degC for 1hour. (25uL reaction)
- Prepared one 2row x 20 lane gel 100mL agarose gel
- Loaded 20uL of digest
- Digest Pattern
- Expected effects
PstI digest
| History | Mutation tube | FRAGMENTS
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 41 | | 8982 | <-
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 42 | | 8982 | <-
|
| pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)-> | 43 | | 8982 | <-
|
| pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)-> | 44 | | 7318 | 2516 | 378 | 105
|
- Results of PstI diagnostics:
EcoRI and BamHI digest (44 with EcoRI only)
| History | Mutation tube | FRAGMENTS
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 41 | | 6871 | 2112
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 42 | | 6871 | 2112
|
| pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)-> | 43 | | 6871 | 2112
|
| pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)-> | 44 | | 10318 | <-
|
HindIII digest
| History | Mutation tube | FRAGMENTS
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 41 | | 4852 | 3479 | 652
|
| pNL29T1-(Comp-g318a)-(GvpL-g351a))-(GvpL-g696a)-(GvpL-g213a)-> | 42 | | 4852 | 3479 | 652
|
| pNL29T1-(GvpL-g351a)-(GvpL-g318a))-(GvpL-g213a)-(GvpL-g696a)-> | 43 | | 4852 | 3479 | 652
|
| pNL26P3-(GvpP-g441a)-(GvpQ-g183a))-(GvpQ-g150a)-(GvpA-t57c)-> | 44 | | 4852 | 3479 | 1335 | 652
|
EcoRI / BamHI digests and HindIII digest
- Results of digests: All look good except 44B which is still being cut ecoRI at t57c (background incomplete digestion of template using dpnI). There does however appear to be a small variation in size but it is hard to tell if this is gel or concentration inconsistancy. 41C HindII 3479 band appears slightly larger than 41A or B.
PstI digest
