I hate studying at 5:30AM

To prepare for downstream processing (i.e., nickel column chromatography), the CISE lab tech needed to prepare 0.5 liters each of the native lysis (i.e., binding), wash, and elution buffers. The concentration of imidazole (MW=68.08 g/mol) in these buffers differs, is as follows: Imidazole concentration in... Native lysis (i.e., binding) buffer is 10 mM Native wash buffer is 20 mM Native elution buffer is 250 mM How many grams of imidazole did she add to the 0.5 liters of lysisbuffer?

.34

To prepare for downstream processing (i.e., nickel column chromatography), the CISE lab tech needed to prepare 0.5 liters each of the native lysis (i.e., binding), wash, and elution buffers. The concentration of imidazole (MW=68.08 g/mol) in these buffers differs, is as follows: Imidazole concentration in... Native lysis (i.e., binding) buffer is 10 mM Native wash buffer is 20 mM Native elution buffer is 250 mM How many grams of imidazole did she add to the 0.5 liters of washbuffer?

.68

The induction culture you watched me prepare in the video had a volume of 1 liter. How much of the 1 M IPTG stock did I add to it to achieve a final concentration of 1 mM?

1 milliliter (ml)

Oh, shoot! Without thinking, you grabbed the wrong micropipetter and used it to add 12 μl of a 50 mg/ml antibiotic stock solution to your 5 ml coli culture. You wanted a final concentration of 100 μg/ml. How much of the LB broth should you add to the 5ml culture to fix the problem?

1 ml

The volumetric amount of plasmid and insert that should be added to a pme-optimized ligation is based upon...

1. The concentration of the prepared DNA fragments 2. The length of each DNA fragment

What happens if there is residual endotoxin in a protein product destined to be a human therapeutic agent?

1. This is bad! If detected prior to product release, the batch will undergo further purification and testing before it can be sold. 2. This is dangerous! If unnoticed and given to patients, those who receive the therapeutic could become very sick or die due to extreme fever.

Which of these is a strategy for making your time spent in the lab more efficient?

1. Use your incubation or spin times to read the next step in the SOP, and/or retrieve, prepare, or label any tubes or plates. 2. When transferring a series of samples from one tube to the next, label and line up your sample-matched tubes so that once you begin, you do not have to look through and find the correct tubes. 3. Process similar samples in parallel.

Number these lab activities according to the order they have been and will be performed.

1. cut, purify, and quantify, the pet duet and synthetic gfp samples 2. ligate pet duet with syn gfp 3. heat shock transform the ligation mixture into the e coli 4. select cells that have taken up a circular pet duet or pet duet (syn-gfp) by plating on a LB+Amp plate 5. screen the isolated colonies to identify those that contain recombinant plasmids

Put these events in the order that they were performed by Dr. Stockwell in her BCA Assay demonstration video.

1. prepare the stock solution 2. aliquot the stock and experimental samples in triplicate into the wells of the microtiter dish 3. prepare the working reagent by mixing solutions a and b 4. add the working reagent to the samples 5. incubate the reactions at 37 degrees for thirty minutes or more 5. measure the absorbance of each sample at 562 nm

According to the BCA Assay Calculation Guide, what is the total volumeof working reagent required for the experiment?

13.8ml

According to the BCA Assay Calculation Guide, what is the required volume of working reagent solution A for the experiment?

13529

According to ligation standards, there should be 100 ng of cut pET-Duet added to the ligation. The cut pET-Duet prep has a concentration of 50.7 ng/ul. How many microliters (uls)of this DNA sample should be added to the ligation?

2 uls

According to my pme calculations, there should be 83 ng of cut syn-gfp added to the ligation. The cut syn-gfp prep has a concentration of 43.8 ng/ul. How many microliters (uls)of this DNA sample should be added to the ligation?

2 uls

Which of these fragments would have the MOST picomolar ends in 1 microgram of DNA?

21 bp primer

Match the final BSA concentration with the microliter volume of 0.5 mg/ml BSA stock mixed with diluent to achieve a final volume of 100 ul.

25ng/ml-5ul 50-10 75-15 100-20 150-30 250-50 400-80

Match the final BSA concentration with the microliter volume of diluent mixed with 0.5 mg/ml BSA stock to achieve a final volume of 100 ul.

25ug/ml-95 50-90 75-85 100-80 150-70 250-50 400-20

According to the BCA Assay Calculation Guide, what is the required volume of working reagent solution B for the experiment?

271 ul

To prepare for downstream processing (i.e., nickel column chromatography), the CISE lab tech needed to prepare 0.5 liters each of the native lysis (i.e., binding), wash, and elution buffers. Her recipe called for a final concentration of 50 mM NaH2PO4(MW=137.99 g/mol) in each buffer. How many grams of NaH2PO4 did she add to each 0.5 liter buffer solution?

3.45

You are setting up a restriction enzyme digest. The enzyme you are using came with its own buffer from the supplier. This buffer is labeled "10x" and the product instructions indicate that you need to dilute it to a 1x working stock when performing a reaction. How much of the 10x stock buffer solution should you add to your 50 μl reaction?

5 microliters (ul)

You are starting a 5 ml coli "overnight" culture. How much of 100 mg/ml Ampicillin stock solution should you add in order to achieve a final concentration of 100 μg/ml?

5 microliters (ul)

To prepare for downstream processing (i.e., nickel column chromatography), the CISE lab tech needed to prepare 0.5 liters each of the native lysis (i.e., binding), wash, and elution buffers. The concentration of imidazole (MW=68.08 g/mol) in these buffers differs, is as follows: Imidazole concentration in... Native lysis (i.e., binding) buffer is 10 mM Native wash buffer is 20 mM Native elution buffer is 250 mM How many grams of imidazole did she add to the 0.5 liters of elutionbuffer?

8.51

To prepare for downstream processing (i.e., nickel column chromatography), the CISE lab tech needed to prepare 0.5 liters each of the native lysis (i.e., binding), wash, and elution buffers. Her recipe called for a final concentration of 300 mM NaCl (MW=58.44 g/mol) in each buffer. How many grams of NaCl did she add to each 0.5 liter buffer solution?

8.77

Which of these is the endotoxin assay most similar to in procedure?

BCA Assay

Which of these describe the steps taken to analyze the result of endotoxin assay results?

Create a standard curve of stock endotoxin solutions. Use the equation of that line to calculate the concentration of experimental samples.

What product attribute is quantified by the BCA Assay?

General Potency (i.e., overall protein concentration)

Suppose that after performing a ligation, transformation, and selection--exactly as we've discussed in this course/project--you get the following results: Experimental Ligation Plates: NO growth Negative control (cells + no DNA): NO growth Positive control (cells + circular plasmid): NO growth Which of these conclusions are consistent with your results?

I may have added too much of the selective agent to the plates. That's right. Maybe you are killing everything because the conditions are just too harsh--even for the resistant cells. I'd go back and check my calculations about how much antibiotic to add to the plates. I may have used plates containing a selective agent that did not match my selectable marker. Yep. Wrong selective agent? Everything will die. Check your plates. The cells may not have been truly "competent" Absolutely. If your cells aren't receptive, they won't take any new DNA in. They could be too old or not prepared properly. I might have made a procedural error in the transformation process. Yep! Too much or too little time at 42 degrees can compromise your transformation rates. Before I can deal with my experimental failure, I first need to figure out why my controls are not working as expected. Absolutely. Don't waste your precious experimental samples until you know that the rest of the procedure and materials are right. Once that gets sorted out, return to your experiment and troubleshoot that, if needed.

If there is found to be residual endotoxin in our batch eluates, what was the most likely source of this contamination?

Leftover E.coli cell wall that remained in the sample after centrifugation of the whole cell lysate

Which of these is the best course of action if you need a cut-site map for an original fragment of DNA (i.e., one that does not come with a commercial map)?

Make your own by cutting and pasting the sequence into a tool such as "NEB Cutter"

How do "native" and "denaturing" buffers used for nickel column chromatography differ in chemical composition?

Native buffers contain imidazole, whereas denaturing buffers do not. Denaturing buffers have more extreme pHs, as compared to native buffers. Denaturing buffers contain urea, whereas native buffers do not.

Which of these key product attributes is the endotoxin assay used to quantify?

Purity--i.e. presence of residual bacterial cell wall components

What is the function of the BSA stock solutions in a standard BCA Assay?

Stock BSA solutions provide the empirical information required to convert measured absorbance to mg/ul protein concentration.

What feature of the engineered GFP allows it to be captured by the nickel affinity column?

The 6x "His-tag", which was engineered to the front of the protein during gene cloning That's right! We gave it this property by cloning the gene into pET-Duet. When doing so, we created a transcriptional and translational fusion with some sequence on the plasmid. That is, when syn-gfp is translated using the promoter, ribosome binding site, and translational start site from the plasmid, it acquires six histidine residues at its front end. This His-tag finds nickel very attractive--it can't resist and will stick to the nickel-coated beads in the column. Don't entirely sure what I'm talking about? Look at the pET-Duet plasmid map to see the His-tag encoding region. Don't have the plasmid map? Do a Google search to find it!

Which of these best describes the way in which the chromogenic endotoxin assay works?

The analyte reacts with a component of LAL, which then becomes activated to cleave the chromogenic peptide. A yellow color is produced.

Suppose you accidentally incubated the cell/ligation mixture for more than 90 seconds at 42oC during a heat shock transformation?

The cells would die and you would get no colonies on your selection plate

nanodrop

This DNA is within the acceptable range for concentration at 45.2 ng/uL. The graph looks good, peaking around 260 nm. The 260/280 is close to 1.8 but low enough to assume some contamination from proteins, the 260/230 is lower than 2 meaning there is contamination from organic compounds and or salt.

Which of these transformed cells should survive and grow on an LB plate containing 100 ug/ml Ampicillin (i.e., Amp100)?

a circular (empty) pet duet plasmid and a circular (recombinant) pET-Duet(syn-gfp) plasmid

quality control goals

analyze batches based on quality and quantity of protein product

How does the plasmid isolation by column miniprep protocol work? Small DNA fragments like plasmids...

are captured by the silica membrane found in the column

Where is the engineered GFP located during the "wash" phase of nickel column chromatography? Engineered GFP is found bound to the column .

bound to the column

Where is the engineered GFP located during the "binding" phase of nickel column chromatography? Engineered GFP is found bound to the column. .

bound to the column.

product development goal

clone the synthetic gene into pet duet and transform the recombinant plasmid into e coli

Where is the engineered GFP located during the "elution" phase of nickel column chromatography? Engineered GFP is found in the solution that drips off the column (i.e., the "flow-through" or "eluate") .

in the solution that drips off the column (i.e., the "flow-through" or "eluate")

commercial manufacturing goal

induce the protein of interest and separate it from all other cellular components

Which of the following is a feature unique to expression (as opposed to cloning) plasmids

inducible promoters

multiple cloning sites contain

many restriction enzyme sites

ladder prediction

sheet 1

Suppose you perform a restriction enzyme-based screen on two potential E.coli/pET-Duet(syn-gfp) clones. To do this you... Grow up liquid cultures using cells from your patched plate. Extract plasmids from both cultures using a miniprep spin column. Set-up EcoRI and AvrII double restriction enzyme digests of both minipreps. Run the digest reaction products on a 1% gel, along with 5 ul of the NEB 1 kb ladder. After doing all this, you get these (black/white inverted) results: Based on these data, which clone is most likely to contain the correct syn-gfp insert?

sheet 1 at the bottom

Suppose you needed 200 ng of pET-Duet to add to a restriction enzyme digest. Your plasmid miniprep concentration is 50 ng/ul and you have just 5 ul left. Do you have enough to complete this reaction, as planned?

yes, at 50ng/ul you have a total of 250ng in your 5ul prep