SBB09Ntbk-Sadao Ota
Sadao 14:02, 27 April 2009 (EDT)
Summary of Activities up to now: * Over the weekend, we got the TBS+AgNO3 assays yielding reddish color for samples 12, 13, 14, 15 * Prepared a repeat of TBS+AgNO3 assay, and left overnight - labelled AG4 Assay V2 * The absorbance readings were inconclusive - the samples have too high background (due to the large presence of E.coli cells) To do today: * Prepare a second round of TBS+AgNO3 assay, under light * Find the absorption wavelengths for the AG4 assay * Quantify the first round of results for the AG4 assay
AG4 Silver biomineralization assay
Preparation of AgNO3 solution
- Weigh AgNO3 powder in an Eppendorf tube (weighed 0.088 grams)
- MW of AgNO3 is 169.87 - dilute 0.088g with 0.500ml offers ~1 M solution
- Make 200ul of 1mM solutions by adding H2O
Preparation of cells for assay
- Spin down 2ml of cells and throw away supernatant
- Add 200 ul of TBS and resuspend cells
- Spin down the cells and throw away supernatant
Running the assay on cells
- Add 180 ul of TBS to each pellet (prepared above)
- Resuspend cells
- Add 20 ul of 1 mM AgNO3 to each TBS/cell-containing tube
- Incubate in the locker overnight at room temperature, according to the paper (ACS Nano, Kitae Nam et. al.)
Determining the AG4 absorption wavelengths
In order to get quantitative data, we need to find out which wavelengths are absorbed by the AG4 peptide silver precipitate. We chose a sample with good colour, and scanned it through the colour spectrum. The instrument used was the Tecan spectrophotometer, and measurements were taken. parameters: 250-800nm range. 10nm intervals. 96 well plate.
- Results were inconclusive
- Control and samples had very similar absorption spectra
- Results probably due to presence of cells
we can get rid of the cells, but need to incubate cells again before that.
Sadao 13:20, 22 April 2009 (EDT)
Summary of Activities: To do today: ==== Preparation of Tris buffer solution ==== * Add 605 mg Tris and 876 mg NaCl to 80 ml H2O * Adjust pH to 7.4 by adding HCl and bring volume to 100 ml ==== Preparation of AgNO3 solution ==== * Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams) * AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution * Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O ==== Preparation of cells for assay ==== * Spin down 2ml of cells and throw away supernatant * Add 200 ul of TBS and resuspend cells * Spin down the cells and throw away supernatant ==== Preparing cells for growth in LBTAg media ==== * Dispense 3ml of LB media into 8 chambers in growth rack * Add 200 ul of Tris buffer solution to the LB media * Add 32 ul of 10 mM AgNO3 to make 0.1 mM LBTAg media * Pick cells from each washed pellet (above) and add to corresponding growth chambers * Cover the rack and incubate overnight at 37C
AG4 Silver biomineralization assay
Preparation of Tris buffer (1*) solution
adjust Ph 7.4 with adding 1M HCl
Preparation of AgNO3 solution
- Weigh AgNO3 powder in an Eppendorf tube (weighed 0.013 grams)
- AgNO3 has MW 169.87 g/mol - dilute 0.013g with 0.750ml for ~10 mM solution
- Make 3ml of 1mM solutions by adding 100ul of 10mM AgNO3 into 900ul of H2O
Sadao 5:04, 21 March 2009 (PST)
I was not in lab today, but Susan and Roger carried out the following work:
Summary of Activities: * Grew one colony from each sample (11-17) and control in 4ml LB media (with CA) * Grew one colony from each sample (11-17) and control in 4ml LB media and 0.1mM AgNO3 (with CA) * [http://openwetware.org/wiki/Image:Silver_binding1.doc growing cells grid]
Sadao 13:41, 20 April 2009 (EDT)
The composite parts have been assembled, and now we are doing assays for the different constructs. I am doing the AG4 silver binding peptide assay (Construct 18) with Carlos, Roger, Dirk, and Susan.
Our assay protocol can be found here:
Silver biomineralization assay
Summary of Activities: * Tested 1M, 100mM, 10mM, 1mM, 0.5mM, 0.1mM AgNO3 in LB media * A precipitate formed with AgNO3 in LB all the way down to 0.5mM * 0.1mM AgNO3 in LB media did not form a visible precipitate * Transformed constructs into bacteria (Roger and Susan) * Plated the cells (Roger and Susan) * We will use constructs 11-17 and a pBca9145-Bca1363 as control To do today: * Test AgNO3 precipitation in LB Media
AG4 Silver biomineralization assay
Preparation of AgNO3 solutions
- Weigh AgNO3 powder in an Eppendorf tube (weighed 0.88 grams)
- AgNO3 has MW 169.87 g/mol - dilute 0.88g with 0.5ml for ~10 M solution
- Dilute it down to 10mM
- Create 1 mM, 0.1 mM, and 0.05 mM dilutions by adding 9+1, 9+1, and 1+1 parts of H2O and AgNO3 solution
Test AgNO3 precipitation in LB media
- Prepare Eppendorf tubes with 200ul of LB media
- Dilute 1M, 100mM, 10mM, 1mM, 0.5mM, and 0.1mM AgNO3 in LB media
- Mix and check for precipitate - if no precipitate, spin down in centrifuge and look for pellet
Sadao Ota 15:03, 9 February 2009 (EST)
My project is SBB09_20154, to make ag43 Display (short) ag43 Display (long) systems. Designed a oligo M10034 to make Autotransporter protein with promotors and so on (corresponding to amino acids 708-1017). Also, designed another oligo M10035 to make Autotransporter protein only (corresponding to amino acids 517-1017).
- Construction files are M10034 M10035
- Oligonucleotide sequences are Oso001, Oso002, Oso003
Sadao Ota 15:03, 16 February 2009 (EST)
Do PCR with the combination of Oso001&Oso002, and Oso002&Oso003.
Sadao Ota 15:03, 23 February 2009 (EST)
Clean up PCR products for gelation analysis
Clean up PCR products, Digest, Clean up and gel again.
Regular Zymo Cleanup (removes the polymerase, dNTPs, buffer, and most of the oligonucleotides from a PCR reaction) here
Load to Gel to perform gelation analysis (dead or alive) here
EcoRI/BamHI Digest of PCR Products (my PCR products are large enough, so no need to modificate the process) here
Sadao Ota 15:03, 25 February 2009 (EST)
Ligation of EcoRI/BamHI digests
I used gel to separate the DNA I need
I also used digested DNA without gelation to do heat shock.
2nd PCR cloning did not work. So, PCRing again...
