IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-19

Site Specific Mutagenesis Attempt 4

Dave and Peng are going to beat this thing until it works.

Vials 8-5, 3-9, 6-4, 10-7, -t for each (dave), 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 8-5, cat gene (dave), negative

#*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

1: ladder
2: 3-9 ZS
3: 3-9 DR
4: 8-5 ZS
5: 8-5 DR
6:10-7 ZS
7:10-7 DR
8: 6-4 ZS
9: 6-4 DR
10:cat ZS
11:cat DR
12:ladder

Miniprep

• The incubations from yesterday were (with help from Ming Ming) miniprepped.
• There were two mistakes with the miniprep:
  • Pipetting the supernatant into the waste when the DNA was in the supernatant
    • Ming Ming helped correct the mistake by re-centrifuging the eppendorf tube with the DNA and extracting more of the supernatant
  • Centrifuging for 1 minute instead of 10 minutes
    • Centrifuged the tubes for another 10 minutes

Sequencing

After the minipreps were complete, one of the tubes that we had used for today was sent off for sequencing.

The two tubes that we are sequencing are:

• Colony 6
• PT (The initial colony)

PCR extraction #5

Samples

• Liquid culture #1 raw (LC1RAW), 50 µL from PCC 7942 flask (2006-7-16) + 100 µL H2O
• Liquid culture #1 purified (LC1PUR), 1 ml from PCC 7942 flask (2006-7-16), spun down for 1 minute at 14k rpm, resuspended in 150 µL H2O
• Liquid culture #2 raw (LC2RAW), 50 µL from PCC 7942 flask (2006-7-7) + 100 µL H2O
• Liquid culture #2 purified (LC2PUR), 1 ml from PCC 7942 flask (2006-7-7), spun down for 1 minute at 14k rpm, resuspended in 150 µL H2O
• Plate #1 (PL1), 1 colony from PCC 7842 streak #1, suspended in 10 µL H2O
• Plate #2 (PL2), 1 colony from PCC 7842 streak #2, suspended in 10 µL H2O

Lyse all samples at 95C for 5 minutes.

Reactions

All reactions are 100 µL.

• LC1RAW and LC2RAW: basically the same as the mega-PCR on 2007-7-10.
  • 10 µL template
  • 10 µL 10x buffer
  • 2 µL 10 mM dNTP
  • 2 µL extF
  • 2 µL extR
  • 1 µL Vent Taq
  • 73 µL H2O

• LC1PUR and LC2PUR: less template
  • 1 µL template
  • 10 µL 10x buffer
  • 2 µL 10 mM dNTP
  • 2 µL extF
  • 2 µL extR
  • 1 µL Vent Taq
  • 82 µL H2O

• PL1 and PL2
  • 5 µL template
  • 10 µL 10x buffer
  • 2 µL 10 mM dNTP
  • 2 µL extF
  • 2 µL extR
  • 1 µL Vent Taq
  • 78 µL H2O

PCR schedule

#94C, 5' note: changed from first protocol to adapt for vent
#94C, 30"
#56C, 30"
#72C, 3.5'
#Cycle to step 2, 7x
#94C, 30"
#55C, 30"
#72C, 3.5'
#Cycle to step 6, 30x
#72C, 5'
#4C hold