Team:WashU StLouis/Notebook

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Notebook

Reading about labwork is almost as fun as doing labwork

June
1 2 3 4
5 6 7 8 9 10 11
12 13 14 15 16 17 18
19 20 21 22 23 24 25
26 27 28 29 30
1 / 30


Stuff that happened on day 1

June 1st
2 / 30


Stuff that happened on day 2

June 2nd
3 / 30


  • Made 1L of LB Media
June 3rd
4 / 30


Stuff that happened on day 4

June 4th
5 / 30


Stuff that happened on day 5

June 5th
6 / 30


June 6th
7 / 30


  • Performed gDNA Extraction on E. Coli MG1655 and Synechocystis 6803
June 7th
8 / 30


  • Designed PCR primers for pgk, pck, fldA, petF, ATP Synthase 1, and ATP Synthase 2
June 8th
9 / 30


Stuff that happened on day 9

June 9th
10 / 30


  • Primers Arrived
  • Ran PCR on ATP Synthase 2 , pgk, fldA, petF, and pdCas9 plasmid
  • Ran all PCR products on gel and recovered ATP Synthase 2, fldA, and petF
  • Ran gradient PCR on pdCas9 plasmid
June 10th
11 / 30


Stuff that happened on day 11

June 11th
12 / 30


Stuff that happened on day 12

June 12th
13 / 30


Made and ran gel for pdCas9

  • Ran PCR on ATP Synthase 1, pgk, and pck
  • Performed gel extraction on yesterday’s ATP Synthase 2
  • Ran pck, pgk, and yesterday’s gradient pdCas9 PCR products on gel and recovered all genes
  • Performed gel extraction on pdCas9, fldA, petF
  • Ran PCR on psc101 and psc102 plasmid
  • Ran psc101 and psc102 PCR products on gel - were recovered
  • Ran gradient PCR on ATP Synthase 1
  • Made agar plates (forgot the antibiotic)
  • Ran golden gate one pot assembly for fldA petF genes and the pdCas9 plasmid
June 13th
14 / 30


  • Ran ATP Synthase 1 PCR products on gel - no bands seen
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Ran PCR on ATP Synthase 1 (3rd times the charm)
  • Ran PCR products on gel - WE SAW BANDS! ATP SYNTHASE 1 RECOVERED
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel Extraction
  • Made new agar plates (with the chloramphenicol)
  • Transformed the petF, fldA plasmids into competent cells
  • Plated colonies on agar plates and set in incubator
June 14th
15 / 30


  • Checked for colonies (both genes were successful)
  • Picked colonies and incubated overnight
  • Ran yesterday’s PCR products on gel - bands were light but all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction
  • Ran PCR products on gel - all were recovered
  • Gel Extraction for pgk, pck, psc101, and psc102 plasmid
June 15th
16 / 30


  • Miniprepped plasmids for fldA, petF
  • Ran sequence PCR on both plasmids
  • Ran PCR product on gel to confirm
  • Clean and Concentrated PCR product
  • Reran PCR on pgk, pck, psc101, and psc102 plasmid because we did not get high enough concentration during gel extraction (for the fourth time)
June 16th
17 / 30


  • Prepared DNA for sequencing
  • Delivered DNA to WashU Med School
  • Added DPnI to psc101 and psc102 gel extract products, incubated for an hour, did Clean and Concentrate, measured conc
  • Ran Golden Gate assembly on pck, pgk, and ATP Synthase
June 17th
18 / 30


  • Electroporation with golden gate products
  • Redid golden gate for pck, pgk, ATP synthase (did not run properly first time)
June 18th
19 / 30


  • Electroporation with better golden gate products
  • Picked colonies for worse golden gate for pck, pgk, ATP synthase
June 19th
20 / 30


  • Miniprep worse ATP, pck, pgk golden gate
  • Seq PCR worse ATP, pck, pgk golden gate
  • Picked colonies for better golden gate for pck, pgk, ATP synthase
June 20th
21 / 30


  • Made frozen stock of better golden gate pck, pgk, ATP synthase
  • Miniprepped better golden gate pck, pgk, ATP synthase (low concentration)
June 21st
22 / 30


  • Ran sequencing PCR for pgk, pck (4 colonies each)
  • Gel Extraction for pgk, pck PCR product (no bands)
June 22nd
23 / 30


  • Ran sequencing PCR for pck and pgk on a gradient
  • Gel Extraction for pck PCR product was successful, not pgk
June 23rd
24 / 30


  • Ran sequencing PCR for pgk on a different gradient
  • Gel Extraction for pgk PCR product was not successful
June 24th
25 / 30


Stuff that happened on day 25

June 25th
26 / 30


  • Picked four new colonies of pgk
  • Started 4 new cultures from frozen stock
June 26th
27 / 30


  • Made frozen stock of the 4 new colonies
  • Miniprepped all 8 pgk cultures and measured concentrations
  • Ran gradient PCR on all 8 pgk plasmids
  • Prepped and sent ATP synthase and pck in for sequencing
June 27th
28 / 30


Stuff that happened on day 28

June 28th
29 / 30


  • Mixed new pgk sequencing primers
June 29th
30 / 30


  • Ran gradient PCR on all 8 pgk plasmids
June 30th
1 / 31


Stuff that happened on day 31

July 1st
2 / 31


Stuff that happened on day 32

July 2nd
3 / 31


Stuff that happened on day 33

July 3rd
4 / 31


4th of July!

July 4th
5 / 31


Stuff that happened on day 35

July 5th
6 / 31


  • Sequencing results for ATP and pck were not good
  • Reran seq PCR on ATP Synthase (bad results)
  • Picked new ATP colonies
July 6th
7 / 31


  • Miniprepped overnight cultures of ATP 5-8 and forgot to make frozen stock
  • Ran PCR on ATP plasmids 5-8
  • PCR for pgk with new primers with gradient
  • Ran pgk PCR product on gels
  • Ran golden gate on pgk PCR gel extract
July 7th
8 / 31


  • Electroporation with golden gate pgk product
July 8th
9 / 31


  • Picked 4 pgk products and inoculated them overnight
July 9th
10 / 31


  • Made frozen stock of new 4 pgk cultures
  • Miniprepped cultures
  • Seq PCR on new cultures
  • All 4 cultures showed correct bands
  • Clean and Concentrate pgk PCR product
  • Prepped pgk for sequencing
July 10th
11 / 31


  • Delivered pgk for sequencing
July 11th
12 / 31


  • New ATP synthase sequencing primers and pck regular primers came in
  • Seq PCR of ATP synthase plasmids with new sequencing primers. No bands
  • Picked 4 new ATP synthase colonies (again)
  • Inoculated MG1655 from frozens stock to make more genomic DNA
July 12th
13 / 31


  • Extracted genomic DNA from MG1655
  • Miniprepped new ATP cultures
  • Ran sequencing PCR on new ATP and got no bands at all. Will use newly transformed cells tomorrow
  • Ran PCR with pck primers, got light bands
  • Reran PCR with pck products
July 13th
14 / 31


  • Ran pck on gel
  • Gel extracted pck and combined gel extracts to improve concentration
  • Golden gate ligation on pck
  • Ran ATP sequencing PCR again with different backbone primers
July 14th
15 / 31


  • Ran gel from yesterday’s ATP PCR. No bands
  • Ran PCR for pfo
  • Incubated pck and BioBrick cells
July 15th
16 / 31


  • Miniprepped pck and biobrock genes (HSP)
July 16th
17 / 31


Stuff that happened on day 47

July 17th
18 / 31


  • Ran ATP seq PCR gradient again. No bands. Done with ATP
  • BioBrick digestion, ligation, transformation with HSP and GFP
  • Ran PCR seq for pck and gel extract
July 18th
19 / 31


  • Delivered pck sequence to med school
  • Ligated backbone for BioBrick
  • Extracted more genomic DNA from MG1655
  • Gradient PCR on pfo
  • Received 𝛥aceE cells from Yale
  • Rehydrated and incubated 𝛥aceE cells
July 19th
20 / 31


  • Ran PCR and gel extract for fldA backbone and petF backbone
  • DPn1 the two backbones
  • golden gate ligation on pfo gene and two backbones
  • Streaked colony from 𝛥aceE plate onto LB/kan plate
July 20th
21 / 31


  • Making media for competent cell protocol
  • Made new LB with MgSO4 for chemical transformation
July 21st
22 / 31


  • Digested BioBrick miniprep to check length
July 22nd
23 / 31


  • Chemically competent cell protocol with 𝛥aceE
  • Miniprepped pfo-petF and fldA-petF
  • Seq PCR miniprepped pfo-petF and fldA=petF
  • Remade LB with MgSO4 with correct concentration
July 23rd
24 / 31


Stuff that happened on day 54

July 24th
25 / 31


  • Restarted comp cell protocol (3rd times the charm)
  • Clean and Concentrate junction 2 for pfo-fldA
  • Conducted HSP test in LB
July 25th
26 / 31


  • Redid HSP test in minimal media M9
  • Recultured BioBrick and DH10B control
July 26th
27 / 31


  • Reset up BioBrick experiment but grew diluted cells with LB instead of M9, so we have to do it again
July 27th
28 / 31


  • Redid HSP test in minimal media M9
July 28th
29 / 31


Stuff that happened on day 59

July 29th
30 / 31


Stuff that happened on day 60

July 30th
31 / 31


Stuff that happened on day 61

July 31st
1 / 31


  • Chemical transformation of pfo-petF, pfo-fldA, petF, fldA into knockout strain, plated 80 and 240 ul, also control
  • Part 1 of interlab study (LUDOX)
  • Transforming 5 interlab plasmids
August 1st
2 / 31


  • Picked and incubated electron donor colonies plated yesterday
  • Picked and incubated interlab colonies plated yesterday (2 per construct)
August 2nd
3 / 31


  • Made frozen stock of incubated colonies and interlab parts
  • Redid BioBrick experiment at 37C

August 3rd
4 / 31


Stuff that happened on day 65

August 4th
5 / 31


Stuff that happened on day 66

August 5th
6 / 31


Stuff that happened on day 67

August 6th
7 / 31


Stuff that happened on day 68

August 7th
8 / 31


Stuff that happened on day 69

August 8th
9 / 31


Stuff that happened on day 70

August 9th
10 / 31


Stuff that happened on day 71

August 10th
11 / 31


Stuff that happened on day 72

August 11th
12 / 31


Stuff that happened on day 73

August 12th
13 / 31


Stuff that happened on day 74

August 13th
14 / 31


Stuff that happened on day 75

August 14th
15 / 31


Stuff that happened on day 76

August 15th
16 / 31


Stuff that happened on day 77

August 16th
17 / 31


Stuff that happened on day 78

August 17th
18 / 31


Stuff that happened on day 79

August 18th
19 / 31


Stuff that happened on day 80

August 19th
20 / 31


Stuff that happened on day 81

August 20th
21 / 31


Stuff that happened on day 82

August 21st
22 / 31


Stuff that happened on day 83

August 22nd
23 / 31


Stuff that happened on day 84

August 23rd
24 / 31


Stuff that happened on day 85

August 24th
25 / 31


Stuff that happened on day 86

August 25th
26 / 31


Stuff that happened on day 87

August 26th
27 / 31


Stuff that happened on day 88

August 27th
28 / 31


Stuff that happened on day 89

August 28th
29 / 31


Stuff that happened on day 90

August 29th
30 / 31


Stuff that happened on day 91

August 30th
31 / 31


Stuff that happened on day 92

August 31st
1 / 30


Stuff that happened on day 93

September 1st
2 / 30


Stuff that happened on day 94

September 2nd
3 / 30


Stuff that happened on day 95

September 3rd
4 / 30


Stuff that happened on day 96

September 4th
5 / 30


Stuff that happened on day 97

September 5th
6 / 30


Stuff that happened on day 98

September 6th
7 / 30


Stuff that happened on day 99

September 7th
8 / 30


Stuff that happened on day 100

September 8th
9 / 30


Stuff that happened on day 101

September 9th
10 / 30


Stuff that happened on day 102

September 10th
11 / 30


Stuff that happened on day 103

September 11th
12 / 30


Stuff that happened on day 104

September 12th
13 / 30


Stuff that happened on day 105

September 13th
14 / 30


Stuff that happened on day 106

September 14th
15 / 30


Stuff that happened on day 107

September 15th
16 / 30


Stuff that happened on day 108

September 16th
17 / 30


Stuff that happened on day 109

September 17th
18 / 30


Stuff that happened on day 110

September 18th
19 / 30


Stuff that happened on day 111

September 19th
20 / 30


Stuff that happened on day 112

September 20th
21 / 30


Stuff that happened on day 113

September 21st
22 / 30


Stuff that happened on day 114

September 22nd
23 / 30


Stuff that happened on day 115

September 23rd
24 / 30


Stuff that happened on day 116

September 24th
25 / 30


Stuff that happened on day 117

September 25th
26 / 30


Stuff that happened on day 118

September 26th
27 / 30


Stuff that happened on day 119

September 27th
28 / 30


Stuff that happened on day 120

September 28th
29 / 30


Stuff that happened on day 121

September 29th
30 / 30


Stuff that happened on day 122

September 30th
1 / 31


Stuff that happened on day 93

October 1st
2 / 31


Stuff that happened on day 94

October 2nd
3 / 31


Stuff that happened on day 95

October 3rd
4 / 31


Stuff that happened on day 96

October 4th
5 / 31


Stuff that happened on day 97

October 5th
6 / 31


Stuff that happened on day 98

October 6th
7 / 31


Stuff that happened on day 99

October 7th
8 / 31


Stuff that happened on day 100

October 8th
9 / 31


Stuff that happened on day 101

October 9th
10 / 31


Stuff that happened on day 102

October 10th
11 / 31


Stuff that happened on day 103

October 11th
12 / 31


Stuff that happened on day 104

October 12th
13 / 31


Stuff that happened on day 105

October 13th
14 / 31


Stuff that happened on day 106

October 14th
15 / 31


Stuff that happened on day 107

October 15th
16 / 31


Stuff that happened on day 108

October 16th
17 / 31


Stuff that happened on day 109

October 17th
18 / 31


Stuff that happened on day 110

October 18th
19 / 31


Stuff that happened on day 111

October 19th
20 / 31


Stuff that happened on day 112

October 20th
21 / 31


Stuff that happened on day 113

October 21st
22 / 31


Stuff that happened on day 114

October 22nd
23 / 31


Stuff that happened on day 115

October 23rd
24 / 31


Stuff that happened on day 116

October 24th
25 / 31


Stuff that happened on day 117

October 25th
26 / 31


Stuff that happened on day 118

October 26th
27 / 31


Stuff that happened on day 119

October 27th
28 / 31


Stuff that happened on day 120

October 28th
29 / 31


Stuff that happened on day 121

October 29th
30 / 31


Stuff that happened on day 122

October 30th
31 / 31


Stuff that happened on day 122

October 31st