Mentors2012

Please Enter your class schedules on the Google Docs Spreadsheet (SchedulingMentorsforGDOcsSp12 )

For Fall 12 Semester:
103112

VDS Mentors are the best!

 * Mentor Tasks** for the new push on protein expression using Sadhana's MtPSTP and Joey and Chrsitina's FtHAP

the students will each do a 500ml expression on their own - but we need to make some reagents for them -- they can see the Targets page for which enzyme they need to do

I have ordered TCEP, PMSF, B-Per, Imidazole, etc. - when these arrive, label them and store appropriately (should say on bottle how to stores, otherwise look up on web) Can see the Solutions & Dilutions spreadsheet in GDocs/Misc/ for some help with calculations and Molecular Weights


 * SOLUTIONS TO MAKE:**
 * LARRY **
 * Make LB** - 2Liters
 * Make 5 M NaCl** - 500 ml in water, sterile filter into a bottle, label, store at Room Temp


 * SADHANA **
 * Make Kanamycin**- stocks 50ug/ul store in -20degC, Make 30 tubes of 500 ul each. __Sterile filter with syringe filter__before aliquoting into tubes. Label. Store in -20degC


 * Make IPTG** - 1 M IPTG stock to make. Make 30 tubes of this with 500 ul in each. Label. Store in -20degC


 * CHRISTINA **
 * Make Lysozyme** - 20 aliquots of 500 ul at 50 mg/ml in 1.7 ml tubes. Label. Store in -20degC


 * Make TCEP -** make ~50 x 100ul aliquots of 500 mM aliquots medium size centrifuge tubes. Label, put in a box and Store at Room Temp in cubby.


 * JOEY **
 * Make PMSF -** 30 x 500 ul aliquots of 10mM in **__Isop____ropanol__** 1.7 ml tubes. Label. store in -20degC


 * Make Imidazole** - 1M stock **make in beaker of 500 ml of water, then bottle top sterile filter it into a bottle, store at Room Temp**

When you work on Friday - work on whatever from above does not get done on Thursday and the Lysis Buffer:
 * JC **

NEW Lysis Buffer for Sonication (make 500 ml)
 * (see GDocs '**Solutions&Dilutions**' in /**Misc **folder for calculations)**
 * Tris 100 mM**
 * NaCl 300 mM**
 * Imidazole 30 mM**
 * pH 8.0 w/ NaOH**
 * label, store at 4degC**

Later:
 * Make Compounds in DMSO** - 50 uM in 100% DMSO, then 5 or 10 uM

Wash Buffer
 * NEW Wash and Elution Buffers and Storage Buffer(see GDocs '**Solutions&Dilutions**' in /**Misc **folder for calculations)**
 * Tris 100 mM**
 * NaCl 300 mM**
 * Imidazole 30 mM**
 * pH 8.0 w/ NaOH**

Elution Buffer
 * Tris 100 mM**
 * NaCl 300 mM**
 * Imidazole 300 mM**
 * pH 8.0 w/ NaOH**

FPLC column buffer - tris Also 'storage' buffer** Tris 50 mM NaCl 150 mM pH 8.0 w/ NaOH Then filter sterilize Then degass

make glycerol stocks of E. coli competent cells (DH5alpha and BL21(DE3) - make plates with no resistance, grow up 4ml culture, see Glycerol Stocks protocol for freezing down. -- there is also a protocol using DMSO in the Molecular Cloning books

Midiprep pGBR22 (for the students first RE digest protocol)

FROM SPRING 2012:

Please Enter your class schedules on the Google Docs Spreadsheet (SchedulingMentorsforGDOcsSp12)

Teaching Points for Labs Spring 12 Larry - create Wikispaces pages for each student for Protein Labs - Done - Thanks! J.C. - '20 questions' form - Done - Thanks! Christina - script for Vina, script to put compound ID label in right place on SDF - Done - Thanks! Michael - pseudoknot stuff, Mini Research Wiki post templates Done - Thanks!

022812

Laptop setup - Michael & Christina - Great job on this guys. A huge help!
Put a tape label on cover of each laptop with name and FRI on it and room # (PAI 2.14). Put a tape label on each mouse and charger (these don't need to be tied to an individual laptop - but just say "FRI PAI 2.14" on them Valine- Dr. B put on ViiA7 and did some Windows updates Aspartate Serine Tryptophan Histidine Methionine no password until you are done installing everything
 * Computer names:**
 * Computer Description:** FRI Laptop
 * __Username:__** VDSclass


 * Set up UT Restricted Wireless - sign into GUEST first then open a webpage to get forced to the Restricted installer
 * Download 'RemoveCredentials.exe' to Desktop
 * Do Windows Updates

Secure Shell - Bevoware Putty Xming - with MESA and Xming Fonts: @http://www.straightrunning.com/XmingNotes/
 * Software to be installed:**
 * Microsoft Forefront Client Security - Bevoware

PyMol - on our Google Docs WinSCP 7-zip Logger Pro ve. 8.3 - Dr. B has USB thumb drive with this http://www.appliedbiosystems.com/absite/us/en/home/support/software/real-time-pcr/ viia7 -software.html
 * Microsoft Office 2007 - Dr. B has CD
 * Viia7 v 1.2 software for RT-qPCR - install software - then get key from Dr. B

Firefox Internet Explorer - Boo! Chrome - Bookmarks to install on browsers: Gmail, UTexas, CNS, FRI, VDS Google Site, VDS Wikispaces, RT-qPCR Course, Residential Halls Study Group, PDB, BRENDA, MolPROBITY

Add password: same as our Google Docs login

Low priority: put an FRI logo as the Desktop. Maybe try this one:

02/26/12: Also, I believe we will be starting the Protein Expression lab early at the end of this week (Thurs & Fri). Basically we will have a few students go this week and just do the Transformation. If you remember - it is a 3 day experiment. So, it is important to spread them out early since we have 35 students! That 1st step is only a transformation - so, you guys have done like a million of those!
 * EXPRESSION LAB:**

Also, I am going to be out of town BOTH this Friday and Next.
 * SCHEDULING EXPRESSION LAB:**

So, for this week on: Thursday - Joey will be in charge of handling the Protein Expression Lab Friday - Joshua will be in charge of handling the Protein Expression Lab

Adam - we might need you to hang around longer on Friday if you can.

Let me know if this does not work and we need to make schedule switches. I will post the lab for the Protein Expression Lab late this week.

Sadhana - I think that you remade the LB -so we should be good to go with that for now.
 * MATERIALS FOR EXPRESSION LAB:**

Larry - I would like to have you check on whether we have enough plates for that lab as an extra task for you this week. --- First check and see what we have. Then make what we are short on. In total we need 35*2 = **70 Ampicillin plates** and **35 No Antibiotic plates**. - Done by Larry - Thanks! Ideally you could do this on Monday or Tuesday. At the minimum, we will need several of each by Thursday.

Thanks, Dr. B

012512- Adam to buy at BioSci storeroom
EDTA (sigma) Cat # E4884 3 medium lab coats (cloth) squirt bottles - 2 of these (for DI water and nanopure water) spray bottles - 2 of these Sharpie pens (black, fine tip) - 6 of these labeling tape (thick and thin) - various colors - maybe about 10 rolls

Larry - did you do a gel of PTP1b or of YopH the other day? - Thanks, Dr. B

Wed Jan 18th
Adam - grow up YopH pNIC-Bsa4 DH5alpha colony in 2x80ml of LB overnight (use water bath shakers since we don't trust the air shaker) Joshua - grow up 2x 25 ml of pGBR22 in BL21(DE3) - use Michael's plate

ToDo List (overall)
Clean out Fridges (silver and white) -- discard old/moldy plates, old tubes Move all Staff plates to the White fridge\ move powdered chemicals in large buckets to bottles generate a mentors hours log sheet generate a time sheets due date sheet (from UT webpage) make name badges and print make -80degC freezer signs (laminate them with new laminator!) make lysozyme - store in freezer make 1M IPTG - store in freezer make Kanamycin stocks 50ug/ul store in -20degC

buy stuff at BioScience storeroom (have to wait til Tuesday to do) bleach alconox?? pasteur pippettes rubber bubls for pasteur pipettes

Monday Jan 16th
Team Building Activity Staff picture Buffer Titration lab (if we have pH probes from other FRI stream) Beer's Law Lab PyMol Labs Purify/Characterize pGBR22 o/n cultures Generate time sheets

Calibrate pH meter: ALL clean glassware: ALL clean out old plates and transfer important ones to white fridge: ALL

Sadhana - Midiprep PSTP pNIC-Bsa4 in DH5alpha Larry - Midiprep YopH pNIC-Bsa4 in DH5alpha

Autoclave colirollers Make LB/Agar plates with NO antibiotic (for sneeze plates) - need about 35 of these
 * divide up colirrolers in to 6 each in sterile tubes of (600 ul tubes) -Done by Joshua

Post RT-qPCR signs in NHB and MBB

//Wolbachia// project Biooscientific project

Sunday Jan 15th
Beer's Law: Christina + Joshua Buffer Titration: none - no probes Transformation of pGBR22 in BL21(DE3) : Michael Grow up pGBR22 in BL21(DE3) overnight - ALL
 * 1) Larry add full labels to Midiprep DNA tubes
 * 2) Sadhana - check protein gel

Sadhana - Friday Jan 13th

 * 1) YopH in BL21(DE3), purify, characterize
 * 2) PTP1b in BL21(DE3), purify, characterize
 * 3) Midiprep - PSTP

Larry (Wed/Thursday)
Express YopH and PTP1B in 1 L of LB each - DONE -- grow to .1 OD then to 0.3 OD (add IPTG) - wait 4 hrs, spin down and store in -80degC Spin down Midiprep DNA colonies that Sadhana grew up (maybe Midiprep them) - DONE

Midiprep (YopH-pNicBsa4, pBGR22) - DONE

Troubleshoot Labs -Wet Labs (Pipetting step of Buffers & Solutions, Beer's Law, Buffer Titration) - **File Not Found**

-Gold labs (if they have been revised by then)

TUES night: Dr. B -
Grow up 2x 80 ml of LB o/n with Kan - for Midiprep (Sadhana- did we use DH5alpha or BL21's for this? - Thanks, Dr. B 1/10/12)

-YopH in pNIC-Bsa4 - J.C. - we have a plate of this as DH5alpha colonies -PSTP in pNIC-Bsa4 - Sadhana - we have a plate of this as DH5alpha colonies -grow up pGBR22 in DH5alpha o/n

Express small culture o/n of YopH and PTP1b

Sadhana - Monday Jan 9th
Make LB media Make some Kan plates, Kan Suc, and Amp

Transformations of plasmids in **DH5alpha** -pGBR22 in pGEM-T - have DNA (use AMP plates)

Transformations of plasmids in (BL21(DE3)) -YopH in pNIC-Bsa4 - J.C.- we have Mini-prep DNA in Final Plasmids box -PSTP in pNIC-Bsa4 - Sadhana - we have Mini-prep DNA in Final Plasmids box -pGBR22 in pGEM-T - have DNA (use AMP plates) -PTP1b in pNIC-Bsa4

Troubleshoot Labs -Wet Labs (Pipetting step of Buffers & Solutions) -PyMol (1 & 2 & 3) - **File Not Found** & **File Not Found**



Mentor Contract on GDocs Timesheet Pay Periods and Due Dates (see bottom of link)



OLD STUFF (Fall 2011):
Tris-acetate buffer that is 500 mM pH 5.5 - see **Solutions&DilutionsforGdocs** spreadsheet

LB media - autoclaved

Make gels

SfApy in pUC19 - Indra SalCA in pNIC-Bsa4 - Krishna MPTPB in pNIC-Bsa4 - Joshua FtHap in pNIC-Bsa4 - Christina
 * Plasmids that we have as Midi Prep**

YopH in pNIC-Bsa4 - J.C.- we have Mini-prep DNA in Final Plasmids box PSTP in pNIC-Bsa4 - Sadhana - we have Mini-prep DNA in Final Plasmids box PP2C in pNIC-Bsa4 - Justin
 * Plasmids that we need to transform (from Mini-prep DNA)**

YopH in pNIC-Bsa4 - J.C. - we have a plate of this PSTP in pNIC-Bsa4 - Sadhana - we have a plate of this
 * DH5alpha colonies that we need to grow up 2x 80 ml of LB o/n with Kan - for Midiprep**


 * Colirollers - autoclave and distribute to small 600 ul tubes (~6 per tube)**


 * RADHIKA: LB/Kan/Suc plates**


 * REAGENTS THAT WE NEED:**
 * For new** Lysis Buffer
 * 50 mg/ml Lysozyme in 500 ul aliquots in 1.7 ml tubes. Store in -20degC**


 * ZOE: TCEP aliquots (already at 500 mM - just move from glass vials to tubes and store at Room Temp in cubby)**


 * YILING: PMSF in 500 ul aliquots in 1.7 ml tubes (10mM concentration in isopropanol) store in -20degC**


 * YILING: 1M Imidazole (make in water) store at Room Temp**


 * ZOE: 5 M NaCl (make in water) store at Room Temp**

Wash Buffer
 * NEW Wash and Elution Buffers and Storage Buffer(see GDocs '**Solutions&Dilutions**' in /**Misc **folder for calculations)**
 * Tris 100 mM**
 * NaCl 300 mM**
 * Imidazole 30 mM**
 * pH 8.0 w/ NaOH**

Elution Buffer
 * Tris 100 mM**
 * NaCl 300 mM**
 * Imidazole 300 mM**
 * pH 8.0 w/ NaOH**

FPLC column buffer - tris Also 'storage' buffer** Tris 50 mM NaCl 150 mM pH 8.0 w/ NaOH Then filter sterilize Then degass