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

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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


 * 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 8-5, cat gene (dave), negative


 * 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: 3-9: 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.



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.

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

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