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

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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.

Label Sample Primer [Template] ng/uL
CY101 HH1 seq_A1 55.75
CY102 HH1 seq_A2 55.75
CY103 HH1 seq_A3 55.75
CY104 HH1 seq_A4 55.75
CY105 HH1 seq_A5 55.75
CY106 HH1 M13F 55.75
CY107 HH1 M13R 35.63
CY201 HH2 seq_A1 35.63
CY202 HH2 seq_A2 35.63
CY203 HH2 seq_A3 35.63
CY204 HH2 seq_A4 35.63
CY205 HH2 seq_A5 35.63
CY206 HH2 M13F 35.63
CY207 HH2 M13R 35.63
CY301 HH3 seq_A1 14.78
CY302 HH3 seq_A2 14.78
CY303 HH3 seq_A3 14.78
CY304 HH3 seq_A4 14.78
CY305 HH3 seq_A5 14.78
CY306 HH3 M13F 14.78
CY307 HH3 M13R 14.78
CY401 HH4 seq_A1 48
CY402 HH4 seq_A2 48
CY403 HH4 seq_A3 48
CY404 HH4 seq_A4 48
CY405 HH4 seq_A5 48
CY406 HH4 M13F 48
CY407 HH4 M13R 48
CY501 HH5 seq_A1 40.65
CY502 HH5 seq_A2 40.65
CY503 HH5 seq_A3 40.65
CY504 HH5 seq_A4 40.65
CY505 HH5 seq_A5 40.65
CY506 HH5 M13F 40.65
CY507 HH5 M13R 40.65
CY601 HH6 seq_A1 33.23
CY602 HH6 seq_A2 33.23
CY603 HH6 seq_A3 33.23
CY604 HH6 seq_A4 33.23
CY605 HH6 seq_A5 33.23
CY606 HH6 M13F 33.23
CY607 HH6 M13R 33.23

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

#*95 °C for 2 min. (melt) 
#*95 °C for 0.5 min (melt) 
#*57 °C for 0.5 min. (anneal) 
#*74 °C for 1.5 min. (extension)
#* Go to step 2 (30x)
#*74 °C for 5 min. (final extension; modified from Silver protocol)
#* Keep at 4 °C forever
Total time:  82 minutes +/- some = 90 min

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

#*95 °C for 2 min. (melt) 
#*95 °C for 0.5 min (melt) 
#*57 °C for 0.5 min. (anneal) 
#*74 °C for 2.5 min. (extension)
#* Go to step 2 (30x)
#*74 °C for 5 min. (final extension; modified from Silver protocol)
#* 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...

SiteSpeific left.jpg

 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
 

SiteSpeific right.jpg

 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