IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-25

  Previous Entry  Current Entry  Next Entry  

Re-innoculation of HH6 from freezer stock
Done at night, 2 re-innoculations off of frozen stock to replenish supply.

Miniprep of reinoculated Hetmann Six
Eluted in 50 uL of H2O. Nanodrop readings:

HH1: 111.5 ng/uL HH2: 47.5 ng/uL HH3: 19.7 ng/uL HH4: 64.0 ng/uL HH5: 54.2 ng/uL HH6: 44.3 ng/uL

Sequencing reactions
We will send out 42 sequencing reactions to GeneWiz tomorrow (6 samples, 7 reactions each). These samples are the Hetmann Six-- Topo + KaiABC (we hope)-- which we have been using in our more recent mutagenesis and splitting attempts.

Transformation
Using the newly arrived Zero Blunt Topo cloning kit, we ligated, transformed, and plated the following:
 * Topo + LC1PUR1 3kb segment (2 plates)
 * Topo + LC1PUR1 400b segment (2 plates)
 * Topo + LC2PUR1 3kb segment (2 plates)
 * Topo + LC2PUR1 400b segment (2 plates)
 * Topo + positive control template
 * Topo + H2O (negative control)

4 µL of template and 20 µL of competent cells were used in each transformation.

Site Specific Mutagenesis Attempt 5
Peng f-ed up the primers...

Dave's template was HH5, Peng's was HH6.

Vials , 3-10, 9-8, 6-7, 5-4, -t for each, cat gene as a positive control.

For cat gene, pbc_ks+, kt_b, kt_bb primers - expected size ~1.1kb

New protocol, based on the Silver protocol on OpenWetWare:

5 µL 10x ThermoPol buffer (Vent) 1.0 µL 10 mM dNTPs 2.5 µL 20 µM forward primer 2.5 µL 20 µM reverse primer 1 µL plasmid DNA 1 µL Vent DNA polymerase 37 uL dH20

PCR Time for 3-9, 6-4, 10-7, cat gene (peng), negative for each


 * 1) *95 °C for 2 min. (melt)
 * 2) *95 °C for 0.5 min (melt)
 * 3) *57 °C for 0.5 min. (anneal)
 * 4) *74 °C for 1.5 min. (extension)
 * 5) * Go to step 2 (30x)
 * 6) *74 °C for 5 min. (final extension; modified from Silver protocol)
 * 7) * Keep at 4 °C forever

Total time: 82 minutes +/- some = 90 min

PCR Time for 6-7, cat gene (dave), negative for each


 * 1) *95 °C for 2 min. (melt)
 * 2) *95 °C for 0.5 min (melt)
 * 3) *57 °C for 0.5 min. (anneal)
 * 4) *74 °C for 2.5 min. (extension)
 * 5) * Go to step 2 (30x)
 * 6) *74 °C for 5 min. (final extension; modified from Silver protocol)
 * 7) * Keep at 4 °C forever

Total time: 111 minutes +/- some = 120min

Results Expected: 3-10: 209bp 9-8: 370bp 6-7: 2402bp 5-4: 80bp

Failed... again...



1: 1 kb+ ladder 2: positive cat dr 3: negative cat dr  4: 6-7 dr  5: 6-7 dr neg 6: 6-7 zs 7: 6-7 zs neg 8: 3-10 dr 9: 3-10 neg dr  10: 3-10 zs  11: 3-10 zs neg 12: ladder

1: 1 kb+ ladder 2: positive cat zs 3: negative cat zs  4: 9-10 dr  5: 9-10 dr neg 6: 9-10 zs 7: 9-10 zs neg 8: 5-4 dr 9: 5-4 neg dr  10: 5-4 zs  11: 5-4 zs neg 12: ladder