Day 1

Miniprep bacteria with pBAD-D4 (name of the plasmid in which we will be inserting the Chitinase genes, tags, etc.) to isolate the pBAD backbone Final DNA concentration: 123.7 ng/μL

Day 2

Restriction digest using BamHI and EcoRI to check for bacterial transformation to check to make sure that the plasmid is the expected length (will also send samples for sequencing) Wells: 1. DNA Ladder. 2. Just EcoRI 3. Just BamHI. 4. No enzyme. 5. Both enzymes

Day 3

Made KPi Buffer… (used in Chitinase Assay)

1. Prepare 800 mL of dH2O in a suitable container.
2. Add 2.405 g of K2HPO4 to the solution.
3. Add 11.73 g of KH2PO4 to the solution.
4. Add distilled water until volume is 1 L.

Day 4

Started Cloning of pBAD-GST-ChiA-FLAG construct. (Function of GST and FLAG: these are protein tags (onto chitinase) to purify and detect chitinase respectively) Did the restriction digest portion, will do gel purification, ligation, and plating tomorrow For protocol click here

Day 5

Ran gel of restriction digest of pBAD only and did Gel Purification (very straightforward after PCR, you want only the copied DNA) of restriction digest of GST/gBlock as we want to preserve the amount of DNA gBlock that we have and will lose less sample via PCR purification. Final concentrations: pBAD: 22 ng/μL gBlock: 10.6 ng/μL Did DNA ligation and plated BL21 competent cells cloned with GST-ChiA full construct (complete protocol is linked in yesterday’s log)

Day 6

Selected 10 colonies from Vector+Insert plate and put in 4 mL of LB+Ampicillin (LB is nutrients for bacterial growth, Ampicillin assists selection of transformed bacteria) media. Incubate in 37 ºC shaker for 24 hrs. Also, did restriction digest and gel on pBAD-D4 vector using MluI and HindIII to check and make sure the enzymes are cutting properly. (If DNA length match expected length, then enzymes are working properly) There is a chance we will have to do ligation and stuff again because there aren’t that many colonies. We will most likely use primers to enhance the gBlock/insert DNA and then try again. Depending on how these colonies turn out after we sequence them. Vector+Insert colony:

Day 7

Did Miniprep of the 10 bacteria colonies that we selected from Wednesday. Used NanoDrop machine to find DNA concentrations of all 10 samples.