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

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Contents

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
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
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


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.

Same as top w/o last lane
Same as top w/o last lane


Miniprep

  • The Hetmann Six incubations from yesterday were miniprepped with help from Mingming.
  • There were two mistakes with the miniprep:
    • Pipetting the supernatant into the waste when the DNA was in the supernatant
      • Mingming 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_Ax_RC). 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 sent 2 samples for sequencing: Hetmann's 6th culture (which turned out well on the digest from 2006-7-18), and our original KaiABC + Topo plasmid that was transformed on 2006-7-12.

Name Strain Primer
CY001Hetmann #6seq_A1
CY002Hetmann #6seq_A2
CY003Hetmann #6seq_A3
CY004Hetmann #6seq_A4
CY005Hetmann #6seq_A5
CY006Hetmann #6M13 forward (to be added by Genewiz)
CY007Hetmann #6M13 reverse (to be added by Genewiz)
CY008TP (from 2006-7-11)seq_A1
CY009TP (from 2006-7-11)seq_A2
CY010TP (from 2006-7-11)seq_A3
CY011TP (from 2006-7-11)seq_A4
CY012TP (from 2006-7-11)seq_A5
CY013TP (from 2006-7-11)M13 forward (to be added by Genewiz)
CY014TP (from 2006-7-11)M13 reverse (to be added by Genewiz)

PCR extraction #5

We attempted to extract KaiABC yet again from cyanobacteria, because so far we've only been succesful once (reaction 21 from PCR extraction #3 on 2006-7-14).

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

  1. 94C, 5' note: changed from previous (Hotstar) protocol, from 15' to 5', to adapt for Vent
  2. 94C, 30"
  3. 56C, 30"
  4. 72C, 3'30"
  5. Cycle to step 2, 7x
  6. 94C, 30"
  7. 55C, 30"
  8. 72C, 3'30"
  9. Cycle to step 6, 30x
  10. 72C, 5'
  11. 4C hold
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