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.