lab practical

accuracy

how close measurment to true value

precision

ability to repeatedly measure a value in fixed situation and get the same results

p-20 pipette

dispensing 2-20 (starts with tens)

p-200 pipette

dispensing 20-200 (starts with hundreds)

p-1000 pipette

dispensing 100-1000 (starts with thousands)

density

mass/ volume

precent error

given- measured/ given x 100

C1 V1=

C2V2

DF=

C1/C2 = V2//V1

SPECTROVIS

• use blank

• cuvette with at least 1mL

Cu=

Cs x Au

As

trendline of serial dilution

y= absorbance

x= concentration

p-value less than .05

significant

temp

location 1- location 2

pH

• pH sensor to channel 1 of lab quest

• invert naglene bottle

• rinse tip of sensor with distilled water

• pace tip 3-4 cm, let stabilize

• begin data collection by pressing play

• 10 seconds collected

• when sampling run complete press square

• analyze , statistics

• average

total solids

• mass of beaker before adding water

• swirl naglene bottles

• use 100 ml graduated cylinder to add 100 mL of sample into each beaker (3)

• oven 100-105 for one week

• measure mass of solids with beaker

• subtract mass of empty beaker from mass with solid

• if solids at least .025 cintune

• average ts mg/L

turbidity

• connect turbidity sensor to lab quest via usb connection, allow warm up for a minute

• turbidity sesor uses plastic cuvette label S, near top

• use pipette bulba nd serelogical pipetete to fill cuvettte with 3 mL of water sample

• invert

• read turbidity in NTU

DO

• plug DO probe to channel 1

• remove case rinse tip, dry

• set switch on probe to collect data in % saturation

• submerge tip to naglene bottle with sample depth of 4-6 cm, metal submerged

• hold 40 sec

• data collection 10 sec

BOD

• fill BOD bottle to neck with sample water

• plug the dissolved ovygen probe into channel 1 of lab quest

• riinse dry

• set switch to collect data in mg/L

• submerge bronle in 4-6 cm hold for 40 sec

• data collection 10 seconxds

• analyze, statisitcs

• place BOD bottles in dark

• record DO in mg/l

nitrates

• attach probe clamp to support stand

• carefully insert nitrate ISE to probe clamp and make sure it was set up vertically

• raise the clamp to allow room for rinsate beaker to be placed under probe

• uncap standerd

• leave probe in standerd for 30 min

• plug sensor to channel 1 of venier interface

• sensors → calibrate → nitrate ISE

• calibrate now

• live voltage stabilize , enter 100, keep

• remove nitrate probe from standerd and add to rinsate beaker

• rinse, dry probe

• live voltage → 1 → keep

• OK to finish calibration

• collect nitrate concentrate data

• add to naglene

• 60 secendonds before data collection

• Nlyze stat

fecal coliform test

• set up 1 DSLB (clear cap long)and 2 SSLB (yellow caps), label

• invert 50 ml conical sample water tube

• with 10 ml serilogical pipette transfer 10 ml of water to DSLB tube

• with p-1000 transfer 1 ml of sample water to other sslb tube

• with p-200 transfer 100 ul of sample water to other SSLB tube

• incubate tube at 44.5 degrees celicus for 24 hours

• then refrigerated till next lab

• record number of tubes with 10% gas or more

• table to determine MPN (# of of three giving poitisve )

• MPN= CFU

colony forming unit

• use to determine amount of substance in colony

• determine what volumes to pipet for a 3x serial dilution in a total vollume of 900 ul

• label 6 microfuges 0-5

• dispense sterile water in each microfuge according to calculations

• invert 50 ml conical tube with water sample. add 900ul water sample to tube 0 per table

• pull your calculated transfer value from tube 0 and add to tube 1

• vortex and continue

• obtain LB plate, draw grid and label on bottom half of petri dish

• most diluted sample in grid 5 and work backward, 10ul spots

• place in refrigerator, count spots

total phosphate

• first digest sample, then use spectrometer to create standard curve

• to prepare digest water sample for testing add phosphate RGT powder to sample flaask

• swril and label SW 5 min

• add 1ml digested water sample to labeled cuvette

• determine volumes to pipet 2x serial dilution in total volume 2 ml

• calibrate spectrovis to measure for absorbance

• go to senors and calibrate and then spectrometer

• find cuvette with blank and press finish calibration, press okay

• sensors to data collection, mode to full spectrum, to time based

• change wavelength 565 press okay, label cuvettes 0-3

• prepare phosphate standerd stock

• add 25ml of 10 mg/L phosphate standard stock solution

• add powder packet swirl 5 min

• find absorbance for each cuvette based on table

• make trendline

DNA barcoding (bacterial inoculation of liquid culture)

• helps to identify organisms

• lab microfuge tube

• add 200 ul of LB medium to microfuge tube

• look at your agar streak plate pick discrete bacterial colony to use

• get a sterile stick from the glass bottle, put stick with colony into LB medium in microfuge tube

• vortex

• ta will incubate

PCR

• denaturation step

  • purified genomic DNA from sample placed in microfuge tube and heated for 1 min at 95

  • makes double to single strand

• primer annealing step

  • use forward and reverse primer, one at 5’ other at 3’

  • thermal cycler drops temp from 95 to 50

  • two primers anneal with the DNA

• Synthesis step

  • temp raised to 72

  • allows taq polymerase to synthesize DNA in 5’ to 3’ direction

  • dna between primers replicated

• master mix

  • made of polymerase dNTPs buffer, forward and reverse primers

  • positive and negative samples

gel electrophoriesis

• setting up and running agrose gel

• agrose melted in buffer to create jelly, cooled then placed in box wiht electrodes and buffer

• blue gel loading dye added to samples (glycerol, xylene cyanol, bromophenol blue)

• dna fragments of known sizes loaded (dna ladder)

• to make DNA visible under UV add ethidium bromide

• 2.0 agrose gel in 1x tae buffer

• fill gel box with buffer

• pull out comb

• load 8up DNA ladder to lane 6

• load 4 ul of S, P, and N

• run at voltage of 175

• take gel to imaging system