DSC SOPs

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Dicty Stock Center SOPs

Stock Center Standard Operating Procedures: Order processing

  1. When an order is placed through the system, an e-mail will automatically be sent to ‘dictystocks@northwestern.edu’.
  2. Enter order into issue tracker:http://bugs.dictybase.org/issuetrackers/SC/
    1. Copy the entire email starting from ‘The following order has been placed…’ This will include the name, address, FedEx number and phone number (needed on the FedEx form).
    2. Some people may choose to pay with a credit card – they will call the lab and we enter the credit card number on the FedEx form.
    3. The Issue number is used as the Order number for our internal tracking.
    4. Order status in the Issue tracker will now show as ‘Open’.
  3. Print order from the email (or from issue tracker). After the order is completed this will be stored in the binder containing orders.
  4. Storage location of the cells; found in the automatically generated order e-mail.
    1. Strains are stored in the liquid nitrogen tanks. There are 3 tanks labeled A, B, and C that are triplicates of each other. Use first from Tank A; if stocks from Tank A run out, use from Tank B; if those run out, use from Tank C. When stocks are only available in Tank C, make new stocks to fill Tanks A and B. Tank C is located in the Olson Building, on the basement level, in the McGaw freezer farm room.
    2. Plasmids are stored in the -80, in racks labeled ‘DSC’ in the third shelf from the top. Plasmid boxes 1 to 15 are in the first rack on the left, boxes 16 and on are in the second rack. Plasmids in those boxes are stored in bacteria. A duplicate of the plasmids is in the chest freezer in the common room.
    3. Plasmids are also stored as DNA. These are located in the DSC -20 freezer (top shelf) in the same room as the hood; they are stored in styrofoam boxes. The list and inventory of the plasmids available as DNA is available in the database.
  5. Label plates: before thawing cells, prepare plates. The easiest is to put the order # (which corresponds to the issue # in the Issue tracker) on each plate. Sometimes (especially when there are many orders at the same time), it’s helpful to also add how many items are in the order (for example, the plate is the 1st out of 5, etc), so that when you ship, you ship every item ordered.
  6. Thawing Dicty cells: Cells are available as either (1) cells*; (2) spores, (3) axenic. We prefer using cells or spores; because for axenic cells we need to use one tube each time a strain is ordered, so stocks run out very rapidly.
    1. Prepare a bucket with dry ice. When you take a tube out of the liquid nitrogen or the -80, place it in the dry ice so it doesn’t thaw.
    2. Recover ‘cells’ and spores using a sterile screwdriver, taking some cells and plating on LPB agar plate with spread with E.coli. This is the default plating condition but klebsiella may also be used along with SM/5 plates.
    3. Axenic cells are recovered by thawing and plating in HL5 on culture Petri dishes. If appropriate, add selection drug the next day.
    4. See also http://dictybase.org/techniques/media/dicty_storage.html#thaw
    5. Note that in large orders, when cells grow at different rates, we can ship in two or more batches, depending on the order size.
  7. Keeping strain inventory to date. The strain inventory lists how many tubes are available for each tube. For ‘cells’ and spores we usually store 1-2 tubes per tank. Axenic strains are stored in triplicates in each tank, so there should be 9 tubes when a strain is first stored. As you use vials, go in the inventory and subtract 1 to the current number of vials (ie, if it says ‘7 vials’, change that to ‘6 vials’). This is helpful because you know which tank to look in: if there are between 7-9 tubes left, they will be in Tank A, if there are between 4-6 vials left, they will be in Tank B; 1-3 are in Tank A. If there are 3 vials left, make new stocks.
  8. Thawing bacteria. Bacteria are in the -80 in racks labeled DSC, in boxed numbered 1-18. Note that there are 81 positions in the boxes (9x9) and that there is one empty in the lower right corner (position 81)- When someone orders more than one plasmid/bacterial strain, we try streaking several on the same plate. You can easily streak up to 4 different bacterial strains on a plate but it is best not to do this with international orders.
  9. Updating the Issue tracker: Change the Order status to ‘Plated (to ship’). This will show up as ‘Cells growing to ship’.
  10. Notes and observations. If you find a strain is not growing well, or is not resistant to the drug markers, or shows a phenotype different from that reported, make a note in the ‘Strain Record’.
  11. Wrapping cells: When cells are ready to ship, insert in small cardboard boxes; more can be ordered from http://www.packagingsupplies.com/cart/mycart.cfm (catalog # C342H6).
    1. Petri dishes: Seal each plate with parafilm and then wrap each plate in paper towels so that they don’t bang against each other. Add additional paper so plates do not move in the box.
    2. Axenic cultures: put 5 ml of culture (at about 1x10e6 cells/ml) in a 15-ml conical (Falcon) tube. Seal the top of the tube in with parafilm; place into a Ziploc bag and then pad with paper as above and put in box.
    3. For all packages, include a copy of the small note in the ‘package slip.doc’ file.
  12. Filling in shipping forms: Most users use FedEx; the easiest for us as we have a fedex DSC account making the process much faster. A copy of the waybill and receipt should be stored in the small filing cabinet behind the desk.
    1. The package needs to be weighed, but for 2 plates it’s about 0.5 lbs.
    2. Declare a value of 10$; make sure to specify the currency USD
    3. Write on the description of goods harmless research.
  13. Information specific for each carrier:
    1. FexEx can be sent from the basement of Terry.
      1. We have FedEx forms, envelopes and boxes in the drawers below the bench (those should be identified with red labels). Remember that domestic and International FedEx forms are different.
      2. Both forms, including the international customs declaration, can now be created online using the DSC fedex account. For international orders, aside from the waybill and sustoms declaration (printed), you need to also include ‘Manufacturer’s declaration’ and ‘Declaration of Biological Shipments’ Forms – both can be found in the DSC folder and printed from the DSC computer.
      3. For Credit Card orders, because online processing needs a billing address, we use paper forms. As a result, you will need to print out and manually fill in the customs forms for these orders; also located in the DSC folder on DSC computer.
    2. UPS: Essentially the same procedure as FedEx, but most reliable option is to call the UPS international number at 800-782-7892 and schedule a pick-up.
    3. DHL: non-existent in the US for domestic shipments. If an international user wants to use DHL, we need to schedule a pick up as with UPS. The DHL phone number is 800-225-5345.
  14. Send an email to the user. We have a standard email found in the DSC folder on DSC computer.
  15. Updating the Issue tracker: Change the Order status to ‘Shipped’.
  16. When users receive the strains, they should send an email. If not, check the FedEx tracking web page and see if the strain has been delivered. Updating the Issue tracker: When the strains have been received, change the Order status to ‘Complete’.

Stock Center Standard Operating Procedures: Strain Deposits and Storage

  • Spores – Prepare 3 mass plates on LBP w/ E.coli and harvest when all are mature. Harvest in about 30 ml HL5, spin once (5 min at setting 4 in tabletop centrifuge), add 4.5 ml cold HL5 to the spore pellet. Keep on ice. Vortex to resuspend the spores and add 0.25 ml of DMSO with swirling. Mix well, and add 1 ml of spore suspension to 4 prelabeled vials of the same color.
  • Cells – Cells are stored if a strain does not make viable spores. Prepare 3 mass plates on LBP w/ E.coli and harvest when the plates are starting to clear (when they have consumed most of the bacteria). Do NOT wait too long. Harvest in about 30 ml HL5, spin once (5 min at setting 1.5 in tabletop centrifuge), add 4.5 ml cold HL5 to the cell pellet. Keep on ice. Vortex to resuspend the cells and add 0.25 ml of DMSO with swirling. Mix well, and add 1 ml of cell suspension to 4 prelabeled vials of the same color. Do NOT vortex cells once the DMSO has been added, they are fragile at this stage.
  • Axenic cultures – Harvest 1-2x108 cells (30 ml at about 5x106/ml), spin once (5 min at setting 1.5 in tabletop centrifuge), add 10 ml cold HL5 to the cell pellet. Keep on ice. Vortex to resuspend the cells and add 0.55 ml of DMSO with swirling. Mix well, and add 1 ml of cell suspension to 10 prelabeled vials of the same color. Do NOT vortex cells once the DMSO has been added, they are fragile at this stage.

Once the cells or spores have been added to the vials tighten the caps well, place the vials in a styrofoam box (dry the vials first), and put the box overnight in the -80°C freezer on top of another box. The vials can be left in the -80°C freezer for a while, but do not pile up too many. The transfer to liquid nitrogen gets compromised when you try to do too many at once.

Spores and cells are transferred one each tanks A, B and C, with the last tube going into the 'Dicty to test' box. One vial of axenic cells is transferred to 'Dicty to test' box, to be used for viability testing, and 3 vials are transferred to each of tanks A, B and C.

Stock Center Standard Operating Procedures: Plasmid Deposits and Storage

  • When received as bacterial transformant - Streak out on LB+drug plate (usually ampicillin, but check the appropriate map). Pick 2 colonies in 4 ml LB+drug, grow overnight at 37°C shaker. The following day, use 1.5 ml of the overnight cultures to make minipreps. To the remaining 2.5mL of overnight culture, add 1 ml of cold 75% glycerol (v/v); vortex gently, and add 1 ml to each of 4 prelabeled vials (plasmid name + date). Use the appropraite minipreps to perform restriction enzyme digests on the plasmids and run on a 0.8% agarose gel.
  • When received as DNA – If plasmid is received on paper, extract in 50-100 μl of TE. Then use a few microliters to transform into competent bacteria (XL10 Gold preferred). If maxiprep is received, use 0.5 μl to transform competent bacteria (XL10 Gold preferred).
  • Storage – Store 2 vials in plasmid box in -80°C freezer 1, and 2 vials in plasmid box in -80°C freezer 2. Store the minipreps in styrofoam boxes at -20°C.
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