George's Research Log

Lab log (Llog [yō'g])

	Monday May 18 2009
TCS/TCC	Ran qRCR with tetC primers/stds. Standards seemed good. m=-3.4 and R2=.995 Had to omit a few standard wells but otherwise triplicates good. Seems dilutions larger than 500X best for amplifying. All other undetectable.
Misc.	Helped Tee with PCR purification. Settled payment issues with Karen
	Tuesday April 21 2009
TCS/TCC	Tried the restriction digest of friday's (4/17/09) plasmid extractions again. Ran a gel of the products. samples are only restriction digests + neg control. Hmm...really don't know what exactly going on here but it doesn't look good. I took another look at yesterday's gel and it actually looks like in one restriction digest well I got a 200bp band. I don't know how I missed it the first time around but I did bring it up with Rad and he suggested it was worth trying to sequence. So I'll try to set that up for tomorrow.
Misc.	Placed orders. Worked up updating chem inventory
	Monday April 20 2009
TCS/TCC	Re-ran gel of friday's restriction digest since it seemed alittle unreliable. samples alternate between plasmid extract and restriction digest. Well this time I definitely got a successful extraction. I talked to Geetika and it looks like my restriction didn't work. So I'll give it another go.
	Friday April 17 2009
TCS/TCC	Retried my plasmid extract from newly plated colonies Talked to Reef and figure the reason I didn't get any extract last time is because I only took one colony. Seems I was way off. This time took a ton of EColi colonies from my plates. Coordinated with Tee and Ever to print out the TCC/TCS poster. Ran gel of plasmid extract and resitriction digest. Alternating samples of plasmid extract and cooresponding restriction digest. I think part of this can be explained in that my wells were particularly shallow and lots of DNA spewed out the top while I was loading. But still alittle strage to have such large bands. Maybe I should run it alittle longer next time??
Misc.	Received packages from Vetmed. Trained Eduardo's undergrad's in DNA soil extraction.
	Wednesday April 15 2009
TCS/TCC	Checked my plates of recombinant EColi and still didn't get very many colonies. Reef says I'll need more for plasmid extraction. So I took large amounts of these colonies and plated them onto fresh LB+Amp plates (they do not contain Xgal-since I know they should all have the plasmid anyways)
Misc.	Placed many orders to Vetmed today Tried to fix the nanopure water system again. After I called Branstead again they said I did have the right cartridges so I put them in again, but things still didn't work. Will be talking to them again tomorrow. Planned a TCS/TCC poster with Inmyoung and Nannan.
	Monday April 13 2009
TCS/TCC	Ran gel of Restriction samples. Hmm...that didn't work. Will need to try the plasmid extract again. Helped Nannan with DNA extraction from soil.
	Thursday April 9 2009
TCS/TCC	Extracted plasmids from newly plated colonies using QIAGEN kit Ran Restriction Digest of samples
	Wednesday April 8 2009
TCS/TCC	Ran gel of yesterday's redo colony PCR Looks good today so will proceed with cloning. Replated all colonies I sampled for colony PCR. Will use these for plasmid extraction.
	Tuesday April 7 2009
TCS/TCC	Ran another gel of m13F/R PCR products I did this again because one possible reason for yesterday's blank gel could be just that I ran it too long. My expected product is at most 400bp and that is clearly didn't make it on the gel yesterday. Alas, still no good. Still didn't get a pos control so this time I'll redo the PCR procedure. Ran new PCR procedure with m13F/R primers.
Misc.	Made more progress on updating chem inventory Talked with Nannan about TCS/TCC set-up for picnic day
	Monday April 6 2009
TCS/TCC	Ran gel of m13F/R gene products to verify tetC incorporation Hmm....no pos control so i'll double check my PCR procedure and try again.
Misc.	Continued to work on figuring out problem with NANOpure water system
	Wednesday April 1 2009
TCS/TCC	Began cloning procedure for tetC gene. Read more about TCS and TCC in the literature.
Misc.	Fiddled with ordering issues and paperwork Finished cataloging another shelf of chemicals
	Tuesday March 31 2009
TCS/TCC	Used PCR Purification Kit to purify 1:10 tetC PCR product Incubated pCR2.1 vector with this purified PCR product in order to begin cloning tomorrow.
Misc.	continued updating chem inventory
	Monday March 30 2009
TCS/TCC	Ran PCR of tetC using same procedure as before. Ran gel of the PCR product. It looks like I have no nonspecific product but the first well sample is good enough.
Misc.	Updated chemical inventory Placed and received orders
	March 16-29 Finals/Spring Break
N/A	Not at work
	Friday March 6 2009
Misc.	Worked with Mike to stock new cabinet wit chemicals
	Monday March 2 2009
TCS?TCC	Ran gel of tetW amplification product from 2/25 looks alright but will need to do gel extraction on these.
	Friday February 27 2009
Misc.	Placed and received orders Made plan for next week.
	Wednesday February 25 2009
TCS/TCC	Ran PCR of Ahjeong's gDNA with tetW primers Used Ahjeongs successful procedure (PCR MM and thermocycler program) for amplifying tetQ for my tetW amplification.
Misc.	Placed and received more orders.
	Monday February 23 2008
TCS/TCC	Met with Kate, Nannan, and Inmyoung to discuss specifications of microcosm experiment.
Misc.	Placed orders.
	Friday February 20 2008
NA	NOT AT WORK (recruitment weekend)
	Wednesday February 18 2009
AbResistance	Running gel from last week's PCR All samples are dilutions of A.S. #4 gDNA. Each dilution doubled and back-to-back, each primer MM alternating. Checked the amplicon size in Aminov et. al 2002 and lowest band for both tetH and tetC match. It looks like my amplification finally worked!
	Monday February 16 2009
NA	HOLIDAY
	Friday February 13 2009
NA	NOT AT WORK (recruitment weekend)
	Wednesday February 11 2009
AbResistance	Made more dilutions of #4 A.S samples (1:1000 and 1:400) since gel from Friday suggested all my current dilutions were still too concentrated. Ran PCR of tetH and tetC (efflux genes) all dilutions of #4 A.S. gDNA modified thermocycler program
	Monday February 9 2009

	Friday February 6 2009
AbResistance	Ran gel from Wed's PCR Samples: #4 1:10, 1:50, 1:100. 1:200, pos, neg controls. Talked with Geetika and Nannan and they said smearing probably because my DNA is still too concentrated. But from this I know my reagents and gDNA are still good at least. Also, next time I'll try using QIAGEN since that helped me get more specific products last time.
	Wednesday February 4 2009
Ab Resistance	Run PCR using 16S primers and thermocycler program Made dilutions of A.S. gDNA #4 Decided to take Nannan's advice and try amplifying 16S gene in order to determine what concentration of gDNA works the best.
	Monday February 2 2009
Misc.	Talked with Orbelina about missing orders and figured out the situation on the Glycyglycine and Geetika's order. Received packages. Ordered vetmed items.
	Friday January 30 2009
AbResistance	Determined new plan for tackling tetH amplification Since my last few PCR's of tetH didn't work, talked with Nannan about different ways to optimize the reactions. She suggesed first making dilutions of my template DNA and trying to amplify the 16S, this way I can narrow in on the right concentration of template I need for amplification. This will also act as a sort of "pos control" verifying that I don't have anything horribly askew with my thermocycler program and MM. Made further dilutions for this plan of action.
	Wednesday January 28 2009
Misc.	Continued working out ordering issues Placed and received orders Worked on figuring out EtBr issues. Calc'd and corrected EtBr concentration label under gel imaging bench. Worked out calculations for proper disposal of extra EtBr solution.
	Monday January 26 2009
Misc.	Worked on ordering complications Located items and placed their orders. Received orders from Orbelina. Worked on following-up with missing orders.
	Friday January 23 2009
NA	NOT AT WORK (recruitment weekend).
	Wednesday January 21 2009
AbR	Talked with Krassi about new ideas to get good amplification.
	Monday January 19 2009
HOLIDAY	HOLIDAY
	Friday January 16 2009
AbR	Ran gel of PCR reactions from 1/14 (sample order: #1 1:1, #1 1:10, #1 1:100, #2 1:1, #2 1:200, #2 1:20, #4 1:1, #4 1:10, #4 1:100) starting to think now that something may be wrong with my DNA templates.... Ran gel of Ahjeong's gDNA samples ONLY (not a PCR rxn) (sample order corresponds to above gel) looks like #4 1:1 and possibly #2 1:1 only samples with real good DNA. need to think about next step
	Wednesday January 14 2009
AbR	Made new dilutions of gDNA samples Ran PCR of gDNA, #1, 2, 4 at different dilutions with tetH primer.
	Monday January 12 2009
AbR	Worked on fixing PCR Amplification since last time it didn't work. Talked with Reef and others and came up with some ideas Placed orders.


	Monday January 5 2009
Misc.	Busy getting things back on track Helped Nannan with ordering primers Received, returned, and ordered packages Autoclaved trash Located my labnotebooks
	Thursday December 11 2008
ABR	Ordered tetW, tetC primers Talked to Mike about results I got yesterday, looks like other than my 10^6 and 10^5 dil stds, my std curve is good (i.e. good slope and R2 values). Calculated values for primer dilutions I need to do and working on calculating proper PCR reaction conditions for tetH/C.
Misc.	Talked with Nannan about her TRFLP results. Looks like her results were very similar to mine (very small and weak signals) We decided perhaps it would be best to test the fluorescence of the primer.0 Received packages. Helped Irina with an order.

	Wednesday December 10 2008

	Friday December 5 2008
ABR	Ran first trial qPCR Only ran standards and NTC's, need to see if these components all still work.
	Wednesday December 3 2008
ABR	Made Dilutions of tetQ primer and std stocks 7 ten-fold dilutions of plasmid standard beginning with 6.2*10^6 (vol ~250ul ea) 10uM solutions of FW/RV tetQ primers (vol=250ul ea)
Misc.	Turned in packing slips to Orbelina Received packages
	Monday December 1 2008
AB Resistance	talked to Krassi about next steps in qPCR calculated vols of solution for creating fresh plasmid standard dilutions and primer dilutions Next time will pipette dilutions and prepare solutions for tetQ qPCR run.
Misc.	Placed orders for lab upkeep. Received packages Talked with Nannan about Lianhua's TRFLP
	Friday November 28 2008-didn't work.
	Wednesday November 26 2008
AB Resistance	ordered tetH primers
Misc.	emptied VAFB MTBE waste containers for Laleh
	Monday November 24 2008
Misc.	received order located Ahjeong's tetQ and ermC primer stocks
	Friday November 21 2008
AB Resistance	Located Ahjeong's box of gDNA
	Wednesday November 19 2008
AB Resistance	Met with Krassi to talk about all the components of AB Project. Determined I need to ID resistance gene which encodes efflux pump. Need to locate Ahjeong's gDNA Need to locate tet and erm primer stocks Looked at the results from my first qPCR with Rad and talked about how to interpret them.
	Monday November 17 2008
Misc.	Placed many orders for the lab.
	Friday November 14 2008
AB Resistance	Rad trained me in the qPCR protocol. Ran 23 wells of PM1 16S gene using Taqman.
	Wednesday November 12 2008

	Monday November 10 2008
AB Resistance	Talked with Rad about first few steps in preparing things for qPCR. Located tetQ F/R primers Identified reaction components and mixture amounts.
Misc.	Helped Geetika with ordering balance calibration PO.
	Monday November 3 2008
Misc.	Placed orders.
Krassi	Attended meeting on AB resistance project.
	Friday October 24 2008
Misc.	Placed orders
	Wednesday October 22 2008
Misc.	Placed orders
	Monday October 20 2008
Misc.	Trained Nannan in using the FastDNA Extraction Kit.
	Friday October 17 2008
Misc.	Placed orders. Talked to Nannan about training on Soil DNA Extraction. Made sure EtBr solution concentration was correct and up to date.
	Wednesday October 15 2008
Misc.	Talked with Mike about organizational ideas for inventory. Worked on inventory. Contacted IDT for status update on Reef's order.
	Monday October 13 2008
Misc.	Trained Smita in chemical inventory and MOBI room duties. Placed orders.
	Friday October 10 2008
Misc.	Began training others to help with chemical inventory.
	Monday October 6 2008
MOBI	Began organization of chemical inventory procedure.
	Friday October 3 2008
Irina	Filter sterilized, transfered, and wrapped in foil Somsomak 2001 media reagents into plastic and glass containers to be frozen at -20C. Put in the freezer in the analytical room. Flushed trace metal solution with N2.
MOBI	Finished up all the rest of the MOBI room duties.
	Wednesday October 1 2008
Irina	Made trace metal solution.
	Monday September 29 2008
Geetika	learned about streaking out white colonies from last week's cloning
Irina	located and recorded molecular weights of trace elements for media located vitamin reagents
MOBI	placed orders
	Friday September 26 2008
Geetika	Began cloning with Geetika
MOBI	performed regular duties received packages placed orders
	Thursday September 25 2008
Irina	Started making media
MOBI	received packages
	Wednesday September 17 2008
Irina's DGGE	Searched for acrylamide gel ingredients. Added Irina-specific DGGE programs to thermocyclers. Ran the gel of 9/11/08 PCR products. samples: #1 (240X), #1 (120X), #65 (240X), #65 (120X), +, + (10X), + (100X), + (1000X), - Ran it at 100V for 50min.
MOBI	finished weekly tasks. placed/received orders.
	Thursday September 11 2008
Irina's DGGE	Since yesterday's gel looked alittle odd, going to try a new approach--dilutions of positive control (in case genomic DNA so concentrated it's allowing for more nonspecific binding to occur) and dilutions of sample DNA (in case other dilutions will work better for amplification). Also, will make sure to run the gel on 100V for about 40min-compared to yesterday's run of 120V for 30min-to get better resolution of ~150bp expected product.
	Wednesday September 10 2008
Irina's DGGE	Ran the gel of the PCR product just to make sure the primers, etc. all worked well. samples: #1 240X, 65 240X, pos. control (PM1 genomic), neg. control (both samples Lianhua's TRFLP DNA dilutions)
MOBI duties	finished up my regular things.
	Tuesday September 9 2008
Irina's DGGE	learned about DGGE, purpose and practice. Performed PCR for initially DGGE test; samples #1, 65 (both Lianhua's 240X TRFLP DNA dilutions), pos. and neg. controls. Ran the gel of the PCR product just to make sure the primers, etc. all worked well. samples: #1 240X, 65 240X, pos. control (PM1 genomic), neg. control (both samples Lianhua's TRFLP DNA dilutions)
MOBI duties	got started on regular things placed an order for Geetika
	Friday September 5 2008
MOBI duties	placed order with Vetmed helped Austin with mobi duties and talked to Inmyoung concerning ordering.
	Thursday September 4 2008
MOBI duties	received orders, returned packing slips to Orbelina.
	Wednesday September 3 2008
Lianhua's TRFLP	Turned in 8 samples (w/ duplicates) to DBS.
	Tuesday September 2 2008
Lianhua's TRFLP	Performed Restriction Digest on samples #1-11, using HhaI enzyme. Ran a gel to confirm digest. samples #1,3,4,5,6,7,9,11 (neg. cntrl)
MOBI duties	Completed my weekly duties Processed an order.
	Thursday August 28 2008
Lianhua's TRFLP	Purified the amplified products via PCR Purification Kit. Ran a gel to confirm I got DNA at the end. samples #1,3,4,5,6,7,9,11 (neg. control)
MOBI duties	Talked with Orbelina about authorization papers. Received orders.
	Wednesday August 27 2008
Lianhua's TRFLP	Ran gel of yesterday's samples to confirm amplification. samples #1,3,4,5,6,7,9,11 Since gel looked rather smeary I performed Rec PCR on these same samples. Ran the gel of Rec PCR products (75ul). samples #1,3,4,5,6,7,9,11 Hurray! Also, made a Excel sheet for QIAGEN Taq calculations.
MOBI duties	ran through regular duties
	Tuesday August 26 2008
Lianhua's TRFLP	After receiving the new 27F FAM primer: Made working solution of 27F Performed initial PCR amplification of 8 test samples (#1,3,4,5,6,7,11)
MOBI duties	Placed orders.
	Thursday August 21 2008
Lianhua's TRFLP	Continued to search for the 27F primer, but eventually just ordered another vial.
MOBI duties	wrapped up some duties with Austin and showed him how to use the 3rd floor autoclave.
	Wednesday August 20 2008
Lianhua's TRFLP	Searched for the 27F primer stock solution
MOBI duties	placed orders
	Tuesday August 19 2008
MOBI duties	Finished regular tasks for the week Trained Austin in Mobi room duties.
Lianhua's TRFLP	Got things ready for beginning the new round of samples.
	Monday August 18 2008
Irina	Synchronized the order forms so now the one on the S: is up to date. Drained and washed the 6/28/2008 MTBE/TBA container in the fume hood.
MOBI duties	Received orders.
	Wednesday August 6 2008
Irina	Acidified MTBE/TBA groundwater containers in fume hood to ~ph7
Lianhua's TRFLP	Ran gel of PCR purified products since the results from the GeneScan run didn't come out properly. samples #1-18, 30-42, 50-51. Again my positive control didn't come out for some reason. I used PM1 genomic extract.
MOBI duties	placed and received orders
Misc.	talked to Angela about TRFLP procedure
	Tuesday August 5 2008
Irina	IC to do list _DONE_ Parafilm plate edges very well _DONE_ Find a nice bubble-padded envelope (can be small); make sure plate is nice and snug in the envelope. _DONE_ Go down to main office before 3 pm (note lunch break 12-1 pm), get shipping label from Corina and attach to envelope; leave envelope for pick up. _799360465268_ Note down tracking number and record here. Thanks!! But of course!
MOBI	finished up regular duties. received packages and placed orders. trained Austin and Inmyoung on using the order form.
	Monday August 4 2008
Lianhua's TRFLP	Perform Restriction Digest on samples 1-58 (with duplicates) using HhaI enzyme. (96 total reactions)
Irina	Located and Streaked Ec0248 Glycerol Stock onto Amp/XGal plate. Labeled as Ec0248 pcrA D.ag #3, and placed it in 37C incubator.

Never mind!

Posted by Kate Scow at 2008-09-11 03:02 PM

ooops George, your newer log was hiding! Now I found it. This is great.

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Never mind!