***Please note that for the 2019-2020 Academic Year we are requesting that schools purchase pipets, tips, and tubes for all of their students to conduct the project at their schools. To help schools determine how much of each supply they will need, along with the costs, we have created a Supply Calculation spread sheet. Please let us know if you have any questions about the spread sheet or which items to order.
The 2019 WSSP will provide reagents, supplies, sequencing and review for the first two rounds of clones prepared by each student at participating schools. Schools that wish to have students analyze Additional clones will need to purchase materials for the analysis and pay for the costs of sequencing, posting, reviewing and submitting the Additional clone sequences to NCBI. Below is our Policy for doing Additional clones.
WSSP Policy for doing Additional clones during the 2019-2020 Academic Year
1. The WSSP will provide the cDNA library plates and primers for school Teams that decide to conduct the Additional experiments. As is customary, requests for cDNA library plates require 2 weeks advance notice.
2. Participating schools will provide all other supplies and reagents that are required to conduct the Additional wet laboratory experiments. See the table and notes below for suggested materials and vendors.
3. All gel pictures and data about the Additional clones must be posted on the school’s Google Docs Clone Report Sheet. Each school is responsible for determining if their Additional clones are adequate for sequencing and should indicate this on their Google Docs Clone Report Sheet.
4. After posting gel pictures and data on the school’s Google Docs Clone Report Sheet, the plasmid DNA minipreps should then be sent to the WSSP project staff at the Waksman Institute, Rutgers University, Piscataway, NJ, where they will be processed for sequencing by Macrogen. Please Note: To reduce costs, clones will not be sent out individually for sequencing but will be batched with other project clones until there is a full tray.
5. The WSSP staff will post and review the sequences obtained from the Additional clones. After these sequences are fully vetted by WSSP staff, they will be submitted by the project staff to NCBI. Please Note: The WSSP is not responsible for poor quality sequences or sequences that cannot be submitted to NCBI.
6. The cost associated with the support provided by the WSSP for Additional clones (processing, sequencing, reviewing, and posting on NCBI) will be $12.50/clone. The cost associated with a reverse sequence for a vetted clone (as described above) will be an additional $12.50/clone. Schools will be invoiced for the services that are associated with analyzing Additional clones after the plasmid DNA minipreps are received by the WSSP staff (Step 4), but before these sequences are posted on DSAP (Step 5).
Below is a list of supplies that are used in the summer Institutes and academic year program to conduct the molecular biology research project that is part of the WSSP. Most of the supplies used by the WSSP have been purchased through VWR because the company provides Rutgers University with discounted prices. Other vendors may have similar supplies. We strongly recommend that you shop around for the best deal. Some vendors may offer donations, so we suggest you call a supplier first, and explain the project.
Reagents and Supplies Used in the WSSP Project
|10 ml Serological pipets, Individually wrapped1||1||89130-898||12-104||P-2837-10|
|10 ml Serological pipets, bulk bag1||1||89130-910||12-112||P-2820-11|
|17X100 Polstyrene sterile culture tubes||1||60818-725||21-129||215090||T-2841-4|
|Miller's LB solid (make up liquid)2||2 ml||97064-108||11-120||616||216700||MBPE-2020|
|Millers LB (in liquid form)||2 ml||101384-874||611||216650||G-3258-1000|
|cDNA Library Plates - Provided by WSSP||1 colony|
|Ampicillin - Provided by WSSP||.002 ml||97061-442||216858|
|Innoculating Loops Sterile3||1||60820-132||215850||J-5112-4|
|Microifuge tubes, clear (MP, digests, PCR, gels)4||5.5||20170-333||24-282||215230||C-3260-1||223-9501|
|Microifuge tubes, yellow (for MP DNA)||1||20170-335||C-3260-Y|
|1-200 ul yellow tips (MPs, digest, PCR, gels)4||22||53509-010||24-151R||215051||P-1231-200||223-9303|
|100-1000 ul Blue tips (minipreps)||4||83007-378||24-161R||215061||P-1231-1000||223-9350|
|1-200 ul tips, reloads||53509-012||24-151RL||P-1235-200|
|100-1000 ul tips, reload||89003-422||24-161RL||P-1235-1000|
|Illustra plasmid Prep Mini Spin Kit4||1 prep||95026-748||83-594|
|Plasmid Miniprep-Classic Kit||1 prep||97064-828||11-308A||732-6400EDU||27104||A1223|
|Ethanol (required for Miniprep kits)||BDH1160-4LP|
|PvuII-HF Enzyme||1 ul||R3151S|
|PCR Supplies 7|
|Illustra Ready to-Go PCR beads 0.2 ml tube w cap||1||89497-136||84-867|
|Primers - Provided by the WSSP|
|Taq master Mix8||12.5 ul||30035-1||42-131||625||C-5052-1||166-5009EDU|
|PCR Tubes , 0.2 ml flat cap||1||89511-270||24-154||642.2||215242||C-3328-1||TFI0201|
|Agarose gel Supplies|
|Agarose (assuming 8 samples/gel)||.15 g||12001-870||20-101||605-100||217080||E-3119-500||161-3100EDU|
|TAE 50X, liquid||20 ml||97063-692||20-193||607XL||219033||MRGF-4210||161-0743EDU|
|TAE 50X, solid||97062-518||0912-2|
|DNA Sizing ladder (Rec Invitrogen9)||20 ul||95057-692||19-111||750||10787-026|
|Loading Dye||5 ul||470105-014||606||218200||K945-5ML||166-0401EDU|
|Powder Free Nitrile gloves||2||32916-670||44-100||774||706335||G-4048|
|DNA Stain - Gel Red or green||89139-134||217305||89139-138||166-5111EDU|
|Ethidium Bromide- provided by WSSP if can use||2001||217422||161-0433EDU|
1. If students are working in groups of four then one pipet can be used to dispense 2.0 ml of LB to 4 tubes.
2. Because many schools do not have access to an autoclave we suggest that schools purchase sterilized LB liquid to grow their Additional cultures. However, to save costs, instead of purchasing liquid LB it is possible to purchase LB powder and make up the liquid LB in batches. This can be done by either filter sterilization (which is expensive) or the LB can be sterilized by heating in a microwave to boiling and cooling three times. After the final cooling, the ampicillin can be added to the LB and stored in the refrigerator.
3. Instead of the sterile disposable sticks, it is possible to buy disposable sterile plastic inoculation loops. Alternatively, metal inoculation loops can be sterilized using Bunsen burners, which can then be used to inoculate the culture.
4. This is the number of tips or tubes that is required for all of the Additional experiments: PCR, DNA minipreps, restriction digests, and agarose gel electrophoresis.
5. The WSSP currently uses Qiagen Plasmid Miniprep Kits to purify the plasmid DNA. You may find less expensive alternatives from other vendors. If you do buy other kits please note the differences in the volumes used for each of the solutions and do not use one set of solutions for another column.
6. The WSSP supplies PvuII-HF from New England BioLabs for the first two rounds. It is strongly suggested that schools purchase the same enzyme for conducting the restriction digests on the Additional clones they analyze. If regular PvuII is purchased from NEB please note that it uses a different buffer!! Please take care to not swap the different buffers with the different enzymes.
7. To save costs, schools may want to omit the PCR screen of the clones. This will save on PCR reagents and one round of agarose gels. The restriction digest will provide the information on the size of the insert, as well as the quality of the DNA prep.
8. In the past, the WSSP used GE Illustra RTG PCR Beads for the PCR. You may find considerably less expensive alternatives, such as using a Taq mix that is available from other vendors. The Taq mix is usually a 2X mix that contains the PCR buffer, Taq enzyme, and dNTPs for the reaction. Two words of caution if you use the Taq mix: 1) Be sure students use the correct size pipetor when they aliquot the mix; 2) Make sure the students do not add anything to the stock tube of the Taq Mix.
9. For consistency, we strongly recommend that schools use the 1 KB DNA ladder marker from Invitrogen for the agarose gel analysis of their Additional clones. If schools do purchase other markers, then please only use them for the analysis of the Additional clones. Gels of the clones from the first two rounds (that are supported by the program) with non-standard markers can not be reviewed by the WSSP staff.