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.

Then we chose a specific restriction enzyme to cut both the original pBAD-D4 plasmid and our constructed plasmid such that we can distinguish between the two based on the length of their base pairs and the number of cuts that are made. We chose EcoRI-HF. This is in order to check that the plasmids that we are using are what we think they actually are. We want to have 250 ng per restriction digest reaction. Add 2 μL of buffer, 1 μL of enzyme, and fill to 20 μL with water. (Always add enzyme last). Then incubate at 37 ºC for 30 mins-1hr.