IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-19: Difference between revisions

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{{IGEM:/Harvard/2006/Cyanobacteria}}
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<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-18 | Previous Entry]]</li>
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-19 | Current Entry]]</li>
<li>[[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-20 | Next Entry]]</li>
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==Site Specific Mutagenesis Attempt 4==
==Site Specific Mutagenesis Attempt 4==
Dave and Peng are going to beat this thing until it works.
Dave and Peng are going to beat this thing until it works.
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*There were two mistakes with the miniprep:
*There were two mistakes with the miniprep:
**Pipetting the supernatant into the waste when the DNA was in the supernatant
**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
***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
**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
***We fixed this by pipetting the liquids from the spin column back into Eppendorf tubes and re-centrifuging
Line 81: Line 94:
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).
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 [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-18#Digest_of_the_topo.2Bkaiabc_clones_and_the_one_we_were_using_the_past_week|digest from 2006-7-18]] and our original KaiABC + Topo plasmid that was transformed on 2006-7-12.
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 [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-18#Digest_of_the_topo.2Bkaiabc_clones_and_the_one_we_were_using_the_past_week|digest from 2006-7-18]]), and our original KaiABC + Topo plasmid that was transformed on 2006-7-12.


{| {{table}}
{| {{table}}
Line 102: Line 115:
| CY007||Hetmann #6||M13 reverse (to be added by Genewiz)
| CY007||Hetmann #6||M13 reverse (to be added by Genewiz)
|-
|-
| CY008||TP (from 2006-7-12)||seq_A1
| CY008||TP (from 2006-7-11)||seq_A1
|-
|-
| CY009||TP (from 2006-7-12)||seq_A2
| CY009||TP (from 2006-7-11)||seq_A2
|-
|-
| CY010||TP (from 2006-7-12)||seq_A3
| CY010||TP (from 2006-7-11)||seq_A3
|-
|-
| CY011||TP (from 2006-7-12)||seq_A4
| CY011||TP (from 2006-7-11)||seq_A4
|-
|-
| CY012||TP (from 2006-7-12)||seq_A5
| CY012||TP (from 2006-7-11)||seq_A5
|-
|-
| CY013||TP (from 2006-7-12)||M13 forward (to be added by Genewiz)
| CY013||TP (from 2006-7-11)||M13 forward (to be added by Genewiz)
|-
|-
| CY014||TP (from 2006-7-12)||M13 reverse (to be added by Genewiz)
| CY014||TP (from 2006-7-11)||M13 reverse (to be added by Genewiz)
|}
|}


==PCR extraction #5==
==PCR extraction #5==
We attempted to extract KaiABC yet again from cyanobacteria, because so far we've only been succesful once (reaction 21 from [[IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-14#Another_PCR_attempt|PCR extraction #3 on 2006-7-14]]).
===Samples===
===Samples===
*Liquid culture #1 raw (LC1RAW), 50 µL from PCC 7942 flask (2006-7-16) + 100 µL H2O
*Liquid culture #1 raw (LC1RAW), 50 µL from PCC 7942 flask (2006-7-16) + 100 µL H2O
Line 177: Line 192:
===PCR schedule===
===PCR schedule===


#94C, 5' ''note: changed from first protocol to adapt for vent''
#94C, 5' ''note: changed from previous (Hotstar) protocol, from 15' to 5', to adapt for Vent''
#94C, 30"
#94C, 30"
#56C, 30"
#56C, 30"
#72C, 3.5'
#72C, 3'30"
#Cycle to step 2, 7x
#Cycle to step 2, 7x
#94C, 30"
#94C, 30"
#55C, 30"
#55C, 30"
#72C, 3.5'
#72C, 3'30"
#Cycle to step 6, 30x
#Cycle to step 6, 30x
#72C, 5'
#72C, 5'
#4C hold
#4C hold

Latest revision as of 09:08, 10 August 2006

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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 

#*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


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


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
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 2006-7-11) seq_A1
CY009 TP (from 2006-7-11) seq_A2
CY010 TP (from 2006-7-11) seq_A3
CY011 TP (from 2006-7-11) seq_A4
CY012 TP (from 2006-7-11) seq_A5
CY013 TP (from 2006-7-11) M13 forward (to be added by Genewiz)
CY014 TP (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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