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

Contamination again in my bands... something went very wrong, and the products are missing. Definately worth discussion tomorrow.

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

Same as top w/o last lane

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 before transferring the supernatants to the spin column
    • We fixed this by pipetting the liquids from the spin column back into Eppendorf tubes and re-centrifuging

Sequencing

We sent off 14 reactions for sequencing. Peng designed the sequencing primers such that we only need to use the primers in one direction (seq_Ax or seq_AxRC). There are 5 such primers, plus 2 for M13 forward and M13 reverse (we don't know which direction KaiABC was inserted into Topo, so we'll try both). Thus there are 7 total primers, which makes 7 reactions per sample. We used two samples: Hetmann's 6th culture (which turned out well on the [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-18#Digest_of_the_topo.2Bkaiabc_clones_and_the_one_we_were_using_the_past_weekdigest|digest from 2006-7-18) and our original KaiABC + Topo plasmid that was transformed on 2006-7-12.

Name	Strain	Primer
CY001	Hetmann #6	seq_A1
CY002	Hetmann #6	seq_A2
CY003	Hetmann #6	seq_A3
CY004	Hetmann #6	seq_A4
CY005	Hetmann #6	seq_A5
CY006	Hetmann #6	M13 forward (to be added by Genewiz)
CY007	Hetmann #6	M13 reverse (to be added by Genewiz)
CY008	TP (from 2006-7-12)	seq_A1
CY009	TP (from 2006-7-12)	seq_A2
CY010	TP (from 2006-7-12)	seq_A3
CY011	TP (from 2006-7-12)	seq_A4
CY012	TP (from 2006-7-12)	seq_A5
CY013	TP (from 2006-7-12)	M13 forward (to be added by Genewiz)
CY014	TP (from 2006-7-12)	M13 reverse (to be added by Genewiz)

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

• LC1PUR1 and LC2PUR1: 1 µL of LC1PUR
  • 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

• LC1PUR10 and LC2PUR10: 10 µL LC1PUR
  • 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

• 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

• -: negative control
  • 10 µL 10x buffer
  • 2 µL 10 mM dNTP
  • 2 µL extF
  • 2 µL extR
  • 1 µL Vent Taq
  • 83 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