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In the meeting with Steve, Mehtap, and John today it was suggested we have a slightly more informative stile of lab protocol readme:

• Summary
• Materials (include Scripts and Input/Output)
• Time Estimate
• Protocol
• Notes (includes To Do’s)

I have some manual protocols (transformation, protein expression) I will convert to markdown in a 'Protein Expression' branch that will follow this format.

The reactions that have been documented seem to have fairly small volumes (15uL for Calhoun and Swartz). We'll have to test to see how well it scales to ~1mL.

Should we add a density meter protocol here?

We need to deprecate the old versions of SOPs in the wiki.

What are our standard Deck Layouts for the EVO?

This may be helpful:
http://www.jove.com/video/50762/protocols-for-implementing-an-escherichia-coli-based-tx-tl-cell-free

There are a number of buffers that we'll need to make for the plasmid prep protocol

We will likely not need huge amount of these buffers, so I'm not sure that the Revo is the best option for right now! Do we have an estimate of time frame for them to implement the small volumes @Lucelenie? Otherwise, these looks like we might be able to make them by hand.

Note there is already this issue: #35

The main thing from my perspective that needs to be updated is:

• adjust label printing so the name is not cut off

This email from @MehtapIsik is perhaps relevant, maybe @jchodera would want this zip file:

In a previous wetlab meeting we decided these were the fields we wanted on the labels in order of priority (ideal to have them all on the label, but if they won't fit we want at least primary and secondary details):

Primary:

• Substance name
• Fill date
• True concentration
• UUID (has to be entered by user manually)

Secondary:

• Supplier
• LOT #
• CAT #

Less important:

• username
• expiration date

I think @steven-albanese also mentioned:

• not using 'safe position' because it is in fact, not very safe.

Anything else?

When John, Erica and I were taking some protein concentrations last week, we thought that the Denovix was not keeping a record of our history after each measurement session and all of our previous history was completely blank on the Data app.

Turns out, it was not an issue actually. All you gotta do is GO to the Data app and manually adjust the first and last day of the measurements you're interested to look at (just put a timeframe), and it will show up everything recorded by the instrument.

Would be useful to make a note of how much TCEP (in g) needs to be added per L of cell-free buffers A and B:
https://github.com/pgrinaway/lab-protocols/blob/master/cellfree.md#buffer-a

OPening this issue to get the ball rolling on how we select primers for the 96-well PCR! Currently I've got the script set up such that we're using 12 primers in one direction and 8 in the other. In our meeting a few weeks ago, we had thought about using a TargetExplorer-like technique to identify boundaries in an informed manner. I'm not terribly familiar with TargetExplorer, so I'm not sure if we can expand it to incorporate information like secondary structure when making choices of boundaries. @MehtapIsik do you have any insight into this?

It ould be nice to make it clear which reference each part of the protocol comes from.

Links to the papers would also be awesome. Is this possible to do through Zotero?

The current Quantos SOPs don't include everything from the wiki.

How is the cell washing done here?
https://github.com/pgrinaway/lab-protocols/blob/master/cellfree.md#preparation-of-harvested-cells

Do we spin down cells in between washes, or use a filter plate to suction out the wash buffer?

Another idea is to buy a commercial S30-lysate kit to give this a spin on kinases before diving in to produce our own S30-lysate in large scale:
https://www.promega.com/products/protein-expression/prokaryotic-cell-free-protein-expression/e_-coli-t7-s30-extract-system-for-circular-dna/?activeTab=0
https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/protein-expression/

Looks like we would get 30 tries for ~$450, which might be worth it.

Lucelenie has a draft of this already, would be good to add to it John's pre- and post-flight checklists and make sure everything is consistent: https://github.com/choderalab/lab-protocols/tree/master/automation-checklists

Here, it says:

• Centrifuge crude lysate at 12,000rcf for 10 min
• Pre-incubate mixture at 37C for 30 min

Do you extract the pellet or supernatant from centrifugation, or do you resuspend after centrifugation?

If the cells are being grown in autoinduction media, is adding IPTG necessary? Or is that a totally separate promoter system?

This EMBL page has more information on preparing S30-lysate with expressed T7 RNA pol.

It's not clear how long the reaction should proceed--180min seems to work for the authors, but there are ways to extend that. I will add some figures from the paper to the document to demonstrate this.

We may need to revisit the DTT/TCEP/3-ME content of Buffers A and B:
https://github.com/pgrinaway/lab-protocols/blob/master/cellfree.md#buffer-a

Eventually, we want to understand exactly what we need in these buffers and why, but I wonder if we want to translate DTT -> TCEP for now (which would appear in both buffers) and keep 3-ME in Buffer A.

However, this website on what should go into protein purification buffers suggests that TCEP may interfere with Ni bead based purification:
http://bitesizebio.com/7893/how-to-design-the-perfect-protein-purification-buffer/

I'm not sure if this means we want it in both Buffers A and B, or just Buffer A.

Other notes:

A nice paper on the stability of TCEP/DTT is found here:
http://protomnis.com/wp-content/uploads/2014/04/TCEP-vs-DTT-publication1.pdf

Notes on preparation of TCEP stock aliquots (if we want to do that) is here:
http://store.p212121.com/TCEP-HCl/