DailySchedule+for+Summer

A tentative schedule of things we will do over the first few weeks. Sadhana, Joshua, Hong, Jeanette, Marshall, Justin, Larry, Joey, Krishna, Christina, J.C., Indra, Benjamin, Vanna, Keyur, Omkar, Sonny

__**Research Presentation Schedule:**__ July 12th: Joey, Indra, Christina July 19th: Christina, J.C., Sadhana, Keyur, Larry July 26th: Krishna, Jeanette,, Marshall August 2nd: Justin, Hong, Ben, Sonny, Joshua August 9th:


 * Instructions**: prepare 3-5 Powerpoint slides to present your work to date
 * **Intro (talk about your disease and what your protein does)**, **Objectives**, **Methods& Results**, **Future Directions**

**NOTE** (as of 07/19/11): use __new grant account number__ for DNA Sequencing jobs (it is already on the form for you in a pull-down list)- otherwise get it from Dr. B or Thao.

__**Friday Aug 12th**__

10:30 - start prepping plate 1 for RT-PCR 12:00 - 2:00 RT-PCR run on 7900-A machine

Set up plate 2 afterwards and run on 7500 machine

**MONDAY - June 6th** 4 plates of LB with Amp Make 250 ml of LB Nanodrop plasmid DNA submit plasmid to DNA sequencing facility (pGFP or pmCherry)

**TUESDAY - 7th** 12:00 Lunch meeting

Make Amp and Kan stocks (50ug/ul) 4 people: Transform a plasmid for Midi-prep (pGBR22) rest: Transformation Efficiency

Paper copy of safety training to Dr. B

**WEDNESDAY - 8th** 10:30 - Life Technologies talk - Nitin Puri - students invited (in Bio 301) - VDSers meet in PAI 2.14 lab 10 minutes beforehand

Check DNA sequence Analyze DNA sequence exercise - in computer lab

5:00 - Krishna and Christina - pick colony of pGBR22 to grow up overnight

**THURSDAY - 9th** 11:00 - Krishna and Christina - spin down and then Midi-prep pGBR22 RE digest and agarose gel check Dilute some primers

Print out safety training, hole punch and place in VDS sign in folder under the 'Safety' tab.

**FRIDAY - 10th** OFF - midiprep?

**MONDAY - June 13th** OFF

**TUESDAY - June 14th**

HSers -
 * lab safety training and printout
 * Micropipettor calibration
 * Nanodrop, submit DNA to sequencing

Transformation of plasmids to make stocks - pUC19, pNIC-Bsa4 (Sadhana, Jeanette, Marshall, Justin) Dilute some primers (Joey, Larry) - see PrimerDilution instructions on GDocs in MISC collection Make Ampicillin and Kanamycin stocks (J.C., Indra) Catch-up (Hong and Joshua)

Krishna, Christina - show Ben, Vanna, Keyur, and Omkar how to do Micropipette calibration, how to do Nanorop and DNA sequencing

Lab Notebook check

4:00 Lab Group Meeting

**WEDNESDAY - June 15th**

HSers -
 * lab safety training and printout
 * RE digest & Agarose Gel
 * Transformation
 * Make LB and plates (if not already)

Target Selections

Grow up plasmids overnight - pUC19 Sadhana, Jeanette

**THURSDAY - June 16th**

11:00 - Midi-prep pUC19 - Sadhana, Jeanette

HSers -
 * lab safety training and printout
 * Anaylyze DNA Sequence
 * Transformation
 * Make LB and plates (if not already)

PCR of pGBR22 Agarose Gel of PCR

Transform pNIC-Bsa4 (Marshall, Justin)


 * FRIDAY - June 17th**

1:00 - Meet in computer lab to do Primer Design

ProtocolPCRforREcloningGREEN.xls OR ProtocolPCRforREcloningRED.xls ProtocolPCR_pLICsequencing ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall10.doc

HSers: lab safety training, RE Digest?, PCR

ProtocolPCRforREcloningGREEN.xls OR ProtocolPCRforREcloningRED.xls ProtocolPCR_pLICsequencing ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Fall10.doc Grow up plasmids overnight pNIC-Bsa4 (Marshall, Justin, Hong, Indra) HSers - PCR, PyMol intro to CA2?
 * Monday - June 20th**

10:30 - Marshall, Indra Spin down and MidiPrep pNIC-Bsa4 Update individual Wikispaces pages Create a Wikispaces page specifically for your target (if you are working in pairs - only create one page and split up the work). Post relevant target info there - see examples already on Targets page. Christina - check in PTP1b culture grew successfully overnight. If not - then do transformation. Midi-prep pUC19 pNIC-Bsa4 (Marshall, Justin) ProtocolPCR_pLICsequencing ProtocolDNASequencingFacilitypNIC-Bsa4_VDS_Summ11.doc
 * Tuesday - June 21st**

1:30 - HSers - Analyze DNA sequence exercise

4:00 Group Meeting Lab Notebook check -
 * Journal Club

Lab notebook check - moved to tomorrow

Joey, JC - overnight growth of pUC19 in DHFalphas Indra, Hong - overnight growth of pNIC-Bsa4 Indra, Hong - Transformation of PTP1b in pNIC-Bsa4 in DH5alphas

10:15 - Midiprep (Joey, JC, Indra) - spin down and save for after Fire Safety Training 11:00 - Fire Safety training 2-4 pm Middle School students for lab demo (2) Sadhana, Larry - Make 1 Molar IPTG, sterile filter, store in -20 degC (make 20 tubes of 500 ul each) - label "1M IPTG" with date Autoclave 4 x 2L flasks for expression 5:00 - Lab Group Meeting Follow up ( cover slides we didn't get to yesterday) 6:00 - Christina, Sadhana, Christina, Jeanette, Marshall pick colony of PTP1b in pNIC-Bsa4 for o/n expression in BL21(DE3) cells in 50 ml of LB Larry, Joshua -overnight growth of PTP1b in pNIC-Bsa4 in DH5alphas (from Indra and Hong's transformation)
 * Wednesday - June 22nd**
 * Lab Notebook check**

10:00 Protein Expression of PTP1b (4 people) - Sadhana, Christina, Jeanette, Marshall
 * Thursday - June 23rd**

~ 5:00 Joshua, Larry - grow up plasmid in bacteria overnight for Midi-prep tomorrow

11:00 Joshua, Larry, Hong - Midiprep of PTP1b in pNIC-Bsa4 in DH5alphas Hong midiprep of pNIC-Bsa4 in DH5alphas
 * Friday - June 24th**

11:00 - Sadhana, Jeanette, Marshall, Christina - sonicate, etc on protein samples

HSers - lab notebook check

Krishna out Ben - off for 3 weeks

1:00 - Sadhana, Jeanette, Marshall, Christina - Protein Purification &
 * Monday - June 27th**

Continue PCRs from last week

Primer design for pNIC-Bsa4 cloning strategy primers with Lagger and Leader - use these:
 * PCR Primer Design for pNIC-Bsa4 Cloning.doc [|ProtocolPCR_PrimerDesign_for_pNIC-Bsa4CloningSumm11.doc]
 * PDF sheet of pNIC-Bsa4 [|pNICBsa4sequencesfasta.txt]

Start Primer Overlap PCR - protocol on GDocs [|ProtocolPCR_PrimerOverlapVDS_Summ11.docx]


 * Tuesday - June 28th**

11:00 - Christina, Josh - make and run SDS-Page protein gels 11:30 - Jeanette and Sadhana - load and run SDS-Page protein gels


 * EVERYONE** - do Primer Design for pNIC-Bsa4 cloning strategy (see Monday's list)

4:00 Group Meeting
 * Journal Club
 * Lab Notebook Check

After Class: Joey, J.C., Larry, Joshua - prepare LB, flasks and grow up overnight culture of protein

10:30 (all day) - Express protein: Joey, J.C., Larry, Joshua
 * Wednesday - June 29th**
 * EVERYONE** - do Primer Design for pNIC-Bsa4 cloning strategy (see Monday's list)

Start cloning of PCR from Primer Overlap into pUC19
 * ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc

12:00 - Lyse and spin down protein: Joey, J.C., Larry, Joshua
 * Thursday -June 30th**

After you have done Overlap PCRs - you can proceed to clonig (be sure to read how long the steps will take so that you can plan it out!) //ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc//

Jeanette, Sadhana, Christina, Joshua - run FPLC on concentrated sample

11:00 - Purify protein: Joey, J.C., Larry, Joshua (and make gels for tomorrow)
 * Friday - July 1st**

That's right - it's time to challenge your lab mates to a friendly game of laser tag! All PAI 2.14 researchers & anyone else who wants to go are invited! Here are the details:

Friday, July 1st 2:30 pm @http://www.blazertag.com/

Price: $7.25 per person per game for a group bigger then 10 Phone: 512-462-0202

1701 W. Ben White BLVD. Austin TX 78704


 * We'll meet in PAI 2.14 at 2:30 & leave shortly after that (~2:40). We can coordinate carpools at that time.*


 * Monday July 4th - OFF**

If you have finished Secondary PCR, then Start cloning (take approximately **6-7 hrs** to do) The first 3 cloning steps need to be done on the same day (Restriction Digest, Ligation, Transformation) Options: A. Start early and finish before class at 4:00 B. Start as usual but time it so that the ligation step (1-2 hrs) is during the class time.
 * Tuesday July 5th**

//ProtocolCloningLigate_REdigest_Transform_pUC19VDS_Summer11.doc//

Joey, J.C., Larry, Marshall - concentrate protein sample and make protein gels

4:00 - Lab Group meeting


 * Wednesday July 6th**

2:00 Joey, J.C., Larry, Marshall - run protein samples on GEL

If you did not get colonies on your plate, you can start working on the Version 2 of the pUC19 Cloning strategy [|ProtocolCloningLigate_REdigest_Transform_pUC19_VDS_Summer11_version2.doc]

The new Journal Club paper is uploaded [|HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf] Joey, J.C., Larry, Marshall - run FPLC on samples
 * Thursday July 7th**

Dr. B out - work on cloning
 * Friday July 8th**

Turn in Lab Notebook to 'Drug Screening Notebooks' drawer in Painer 2.14 Lab - for check by Dr. B

Day 1: Day 2: Day 3: Day 4: Day 5:
 * Digest, ligate, transform (long day)
 * Grow up colonies
 * -- (simultaneously start alternative strategy)
 * Mini prep colonies and send to sequencing
 * Wait
 * Get sequencing results


 * Monday July 11th**

Need lab notebooks from: Christina, Larry, Justin, Joshua

Update your Wikispaces research page to show latest gel images.

Verify genes within pNIC-Bsa4 by checking your DNA sequencing results of pLIC primers on pNIC-Bsa4

afternoon (~1 ish) Joey, J.C., Larry, Marshall - Test (the other guys') PTP1b in enzyme test Jeanette, Sadhana, Christina, Joshua - Test your PTP1b in enzyme test See protocol on Google Docs

5:00 - **Justin, Indra, Hong, Krishna -** grow PTP1b protein overnight in small flasks

10:00 -**Justin, Indra, Hong, Krishna -** express PTP1b protein
 * Tuesday July 12th**

4:00 Group Meeting and Journal Club (Jeanette's TGF-Beta paper) + Larry Research Presentation: **Joey & Indra** The new Journal Club paper is uploaded [|HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf]


 * Wednesday July 13th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein sonication, purification


 * Thursday July 14th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein making gels, running gels

Day 1: Day 2: Day 3: Day 4: Day 5:
 * Friday July 15th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein FPLC purification
 * Digest, ligate, transform (long day)
 * Grow up colonies
 * -- (simultaneously start alternative strategy)
 * Mini prep colonies and send to sequencing
 * Wait
 * Get sequencing results


 * Monday July 11th**

Need lab notebooks from: Christina, Larry, Justin, Joshua

Update your Wikispaces research page to show latest gel images.

Verify genes within pNIC-Bsa4 by checking your DNA sequencing results of pLIC primers on pNIC-Bsa4

afternoon (~1 ish) Joey, J.C., Larry, Marshall - Test (the other guys') PTP1b in enzyme test Jeanette, Sadhana, Christina, Joshua - Test your PTP1b in enzyme test See protocol on Google Docs

5:00 - **Justin, Indra, Hong, Krishna -** grow PTP1b protein overnight in small flasks

10:00 -**Justin, Indra, Hong, Krishna -** express PTP1b protein
 * Tuesday July 12th**

4:00 Group Meeting and Journal Club (Jeanette's TGF-Beta paper) + Larry Research Presentation: **Joey & Indra** The new Journal Club paper is uploaded [|HerschhornHIVreversetranscriptaseVirtualScreenJMedChem2008.pdf]


 * Wednesday July 13th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein sonication, purification


 * Thursday July 14th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein making gels, running gels


 * Friday July 15th**
 * Justin, Indra, Hong, Krishna -** work on PTP1b protein FPLC purification

__**Monday July 18th**__

Need lab notebooks for last time from: Larry, Justin, Joshua Update your Wikispaces research page to show latest gel images.


 * Justin, Indra, Hong, Krishna -** work on PTP1b protein: make gels, run gels, stain and then destain overnight, concentrate protein,

Thao - prep 2x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday

__**Tuesday July 19th**__


 * reconcentrate protein after gel

4:00 Group Meeting and Journal Club **Hong & Indra** (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer) Research Presentation: **J.C., Sadhana, Keyur**

__**Wednesday July 20th**__

2-4 pm: Middle Schoolers 'shadowing' in lab

Journal Club **paper for Joshua is uploaded** (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer)

__**Thursday July 21st**__ 10:00 -5:00 - **RT-qPCR workshop**: 1st Group: **Sadhana, Jeanette, Joey, Krishna, J.C., Larry**
 * Next week: 2nd Group: **Justin****, Joshua, Hong, Marshall, Indra**
 * Justin, Indra, Hong, Krishna -** run PTP1b protein on FPLC with Dr. B and Thao

__**Friday July 22nd**__

1:30 - **Analyze Data from RT-qPCR workshop**: 1st Group: **Sadhana, Jeanette, Joey, Krishna, J.C., Larry**


 * ~1:00 Justin, Indra, Hong, Krishna -** run PTP1b protein on FPLC with Dr. B and Thao

__**Monday July 26th**__

10:00 am -6:00 pm - **RT-qPCR workshop**:**n** 2nd Group: **Justin****, Joshua, Hong, Marshall, Indra ...... (the new cDNA came in - so we are on for Monday)**


 * Justin, Indra, Hong, Krishna -** PTP1b enzyme activity test

???Thao - prep 2x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday

__**Tuesday July 27th**__

3:00 Justin - FPLC on PTP1b protein sample ??

4:00 Group Meeting and Journal Club **- Joshua** (paper copies of the journal articles are in the lab - in the Drug Screening Notebooks drawer) Research Presentation: **Krishna, Jeanette, Joshua, Marshall**

__**Wednesday July 28th**__

????2-4 pm: Middle Schoolers 'shadowing' in lab

__**Thursday July 29th**__

__**Friday July 30th**__

NEXT WEEK:

__**Monday Aug 1st**__

__**Tuesday Aug 2nd**__ Thao - prep 4x Restriction Enzyme digest of pGBR22 for Middle Schoolers on Wednesday (enough for this week and next time)

Research Presentation: **Justin, Hong, Ben, Sonny, Joshua**
 * 4:00** Group Meeting

__**Wednesday Aug 3rd**__

Thao - prep 2 TAE gels also 2-4 pm: Middle Schoolers 'shadowing' in lab

__**Thursday Aug 4th**__

__**Friday Aug 5th**__ Last day of summer research

NEXT WEEK: nothing ........