Team:U of Guelph/Notebook
Notebook
Experiment Overview
Here we will include a day by day entry for our project. We need to make sure we atleast include the days we did which experiments but more information would be good too. Note who participated in what should be included.
June 5
LB and LB Kan Plates were poured.
June 6
Re-streaked Ecoli BL21, DH5α, and DH5α/ pET-28a from the stocks we were provided.
June 7
Liquid cultures of DH5α, DH5α/ pET-28a and BL21 were inoculated and grown for 48 hours
June 9
Glycerol stocks were prepared from the liquid cultures
June 14
Ordered frc and oxc Full length Sequences
June 19
Ordered gBlock fragments of frc and oxc for use in a gibson assembly
June 20
Ordered original PCR primers to add PstI to pET-28a
June 21
A half sleeve of each chloramphenicol and Kanamycin plates were poured, DH5α/pET-28a was re-streaked, and a DH5α liquid overnight culture was prepared.
June 22
DH5α competent cells were made. Experiment failed at the final step due to forgetting the glycerol before putting cells in liquid nitrogen.
June 25
Ordered primers to add frc and oxc ends to pET-28a(PstI).
July 5
Miniprep DH5α/pET-28a, and PCR to add PstI to pET-28a.
July 6
Gibson Assembly transformation.
July 10
Made LB broth and an agarose gel.
July 12
Re Streaked BL21 (onto LB) and DH5α/pET-28a (onto LB Kan).
July 13
Overnight liquid cultures were prepared of BL21 and DH5α / pET-28a in 4 mL LB broth in plastic culture tubes. Kanamycin was forgotten in pET-28a culture. Ordered Primers for frc and oxc genes without RE end sites.
July 14
Prepared BL21 Competent Cells, Miniprep of DH5α / pET-28a.
July 19
Inoculation of DH5α/pET-28a overnight liquid culture incubated at 37 degrees with shaking.
July 20
Miniprep of DH5α/pET-28a liquid culture.
July 21
PCR
July 24
PCR of pET-28a to add PstI RE site using incorrect Primers
July 28
PCR of pET-28a to add PstI RE site using incorrect Primers
July 29
Gel of frc and oxc PCR
August 3
pET-28a (PstI) PCR
August 8
Ordered New PstI Primers
August 10
PCR of pET-28a to add PstI cut site using new primers
August 15
Agarose Gel
August 17
Ordered New frc and oxc Primers
August 19
Transformation of pET-pstI (66.9 and 68.9) into DH5α. Incubated plates all weekend.
August 21
Re Streaked colonies 1-10 from pET-28a (PstI) transformation plates.
August 22
Final PCR of frc (47.0, 47.7, 49.1 and 51.2) and oxc at (53.8, 55.8, 57.1, 58.0) Liquid cultures of colonies 1-6 from plates. Purification of frc and oxc PCR products.
August 23
Gel of frc and oxc PCR reactions. Miniprep of DH5α pET-28a(Pst1). RE digest pET-28a(Pst1) with PstI and AnaI. Gel of RE digest DH5α pET-28a(Pst1) with PstI and AnaI. RE digest pET-28a(Pst1)(2) EcoRI and PstI, and frc (47.0 and 49.1) and oxc (53.8 and 55.8). Ligation of pET-28a(Pst1)(2) with frc and oxc. Transformation of DH5α with ligation mixtures, +ve control pET-28a(PstI) (2) and -ve control RE digested pET-28a(PstI) (2)
August 24
Gel of ligation mixtures after transformation failed Transformation of DH5α with ligation mixtures, +ve control pET-28a(PstI) (2) and -ve control RE digested pET-28a(PstI) (2) Plated on Kan plates and incubated overnight at 37 degrees.
August 25
Put plates in fridge to store until monday.
August 28
Re-streaked pET-28a(PstI) (2) to obtain isolated colonies. Suspended selected colonies in 10 ul of water. Colony PCR and agarose gel. Liquid overnight cultures of DH5α/pET-28a(PstI)frc (colony 14 and 17) and DH5α/pET-28a(PstI)oxc (colonies 1, 3 and 5)
August 29
Re-streaked pET-28a(PstI) (2) to obtain isolated colonies. Made new sterile LB. Minipreped DH5α/pET-28a(PstI)frc (colony 14 and 17) and DH5α/pET-28a(PstI)oxc (colonies 1, 3 and 5). RE digested DH5α/pET-28a(PstI)frc (colony 14 and 17) and DH5α/pET-28a(PstI)oxc (colonies 1, 3 and 5) with EcoRI and PstI. Made glycerol stocks of DH5α/pET-28a(PstI)frc (colony 14 and 17) and DH5α/pET-28a(PstI)oxc (colonies 1, 3 and 5) and stored in Tetlow lab -80 freezer
August 30
Dry autoclave run to sterilize tips and tubes. RE digested PSB1C3 with EcoRI and PstI. Ligated frc (47.0 and 49.1) and oxc (53.8 and 55.8) into PSB1C3. Transformed PSB1C3frc and PSB1C3oxc into DH5α and plated on Kan plates, +ve control pET-28a (PstI), -ve control RE digested PSB1C3. Liquid culture of pET-28a(PstI) (2)
August 31
Realized PSB1C3 carries Chloramphenicol resistance and transformations need to be redone.
