Team:HK CPU-WFN-WYY/Notebook


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Notebook

DNA no. Abbreviation Parts registry ID
DNA 1 Piri6 (PiSp_Trichonephila clavipes_6) BBa_K3816009
DNA 2 Masp2_2 BBa_K3816015
DNA 3 Piri2 (PiSp_Trichonephila clavipes_2) BBa_K3816005
DNA 4 Masp2_1 BBa_K3816014
DNA 5 Piri5 (PiSp_Trichonephila clavipes_5) BBa_K3816008
DNA 6 Masp1_102 BBa_K3816011
DNA 7 Piri4 (PiSp_Trichonephila clavipes_4) BBa_K3816007
DNA 8 Tusp (Tubuliform silk_Nephila Antipodiana) BBa_K3816002
DNA 9 Masp1_103 BBa_K3816001

June

  1. Literature review
  2. programmatically finding target sequences from NCBI
  3. manually screening sequences, creating gene constructs and primer pairs (for screening and PCR) in Benchling
  4. order reagents

1/8-7/8

  1. Test amplification of target genes by low-fidelity DNA polymerase of DNA 1-9

8/8-14/8

  1. Test amplification of target genes by low-fidelity DNA polymerase of DNA 1-9
  2. DNA amplification by high-fidelity DNA polymerase of DNA 1-9

15/8-21/8

  1. 1st Digestion of DNA1,2,3,4,6,7,8,9 with Sac I and Sal l
  2. Ligation of DNA 1,2,3,4,6,7,8,9
  3. Transformation of DNA 1,2,3,4,6,7,8,9
  4. Incubation overnight
  5. colony PCR
  6. DNA amplification by high-fidelity DNA polymerase of DNA 1-9

22/8-28/8

  1. 2nd Digestion of DNA 1,2,3,4,6,7,8,9 with Sal1 and Sac1, plasmid
  2. Ligation
  3. Transformation
  4. Miniprep for plasmid (transformation and streak plate)
  5. Incubation
  6. Run gel
  7. Gene clean
  8. Colony PCR
  9. cell culture
  10. Miniprep to extract plasmid (TR -ve)

29/8-4/9

  1. Colony PCR
  2. run gel
  3. LB culture
  4. miniprep

5-11/9

  1. Colony PCR and gel electrophoresis
  2. 1st digestion of DNA 1,2,3,4 with Sac1
  3. Gene clean of DNA 2,3,4 without gel
  4. 2nd digestion of DNA 1,2,3,4 with Sal1
  5. Run gel (agarose) of DNA 1,2, 3, 4, plasmid and ladder to check digestion product
  6. gene clean with PE and PW wash
  7. Ligation of DNA 1,2,3,4 (DNA2,3,4 overnight)
  8. Transformation DNA1
  9. PCR DNA 8,9

12/9-18/9

  1. Double digest (DNA 6-9+Plasmid)
  2. Run gel (DNA 6-9+Plasmid)
  3. Gene clean with PE and PW wash of plasmid
  4. Ligation DNA 2-4,6-9(DNA 2-4 with mighty mix)
  5. Transformation of DNA 2, 3, 4, 6, 7, 8, 9 and plasmid
  6. Spread plate with DNA 2,3,4, 6, 7 or plasmid for each plate
  7. Prepare competent cells of E.coli DH5a

19/9-25/9

  1. Check colonies 6,7(DNA 6:failed, DNA 7:positive but colony too small)
  2. Mini. prep
  3. Run gel
  4. Ligation, transformation and spread plate using old stock
  5. Colony PCR of DNA 3, 7with F primer and R primer
  6. Incubation
  7. Checking the competent cells by transformation
  8. Prepare competent cells of E.coli DH5a and E.coli LMG194

26/9-2/10

  1. Digestion DNA1,7,8,9 and plasmid
  2. Gel electrophoresis
  3. Ligation
  4. Testing PCR
  5. Cell culture
  6. Miniprep 1,2 (DNA 7)

4/10-10/10

  1. Double digestion with gene clean without gel(DNA 2,3,4)
  2. Gene clean of without gel eluted with new elution kit with smaller product volume(DNA 2,3,4)
  3. Ligation using new ligase and buffer(DNA 2,3,4)
  4. Transformation (DNA1,2,3,4,8,9) and spread plate
  5. Colony PCR(DNA 1,8,9)
  6. PCR screening of 15 colony, 5 in each plate, with FWD primer and REV primer(DNA2,3,4)
  7. Gel electrophoresis of DNA 1,8,9,
  8. Liquid culture (recombinant plasmids transformants)
  9. Re-digestion of DNA 1,8,9(Sac1 and Sal1)2,3,4(using XbaI, HindIII of miniprep product from some colonies to check if there is insert)
  10. Miniprep
  11. Liquid culture and miniprep (to increase concentration of plasmid)
  12. Run gel (DNA 7 miniprep product)

12/10

  1. Sending miniprep products to sequencing company

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