Wet laboratory

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Mol.biology // Next-generation sequencing

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Содержание


Common laboratory questions/mistakes.
NB! This material is for experienced laboratory stuff, NOT for studying of basic laboratory techniques.


[править] Clean work

The meaning of the term "clean work" is strongly dependent on the application. It is necessary to understand

» what is the aim of the work
» which contaminations should be avoided
ApplicationMain contaminationsMain precautions
Eucaryotesbacteria, fungi, other strains of eucaryotic cells
  • separate room with special rules
Bacteriaother bacteria with the same antibiotic resistance
  • wash bench
  • sterilize bench, hands/gloves and instruments with Ethanol
  • use autoclaved plastic and media
  • special container for garbage
Library preparationother libraries
  • set up "pre-PCR" and "post-PCR" areas
  • aliquot reagents
  • wear gloves
  • do not prepare libraries from highly different amount of starting material in parallel
  • clean electrophoresis unit/plates and gel-cutting place immediately after use
  • be careful with "common"
» equipment
» PCR primers
PCRmaterial from previous amplifications
RNARNases, skin, saliva, dry bacterial media
  • wear gloves
  • do not store RNases with other reagents
  • weight solid RNases under fume hood
  • etc.
Enzymatic reactionsdetergents, dirty plastic, haotropic salts
  • do not store RNases and Proteinases with other reagents
  • weight detergents, RNases, Proteinases under fume hood
  • use autoclaved plastic only when it is really necessary
  • do not use DEPC-treated water


NB! rules and techniques of "clean work" are not complex. It is not a problem to work clean for 15min. The problem is, that it is necessary to work clean always, independent of your state of mood and health. If contamination happends, it is difficult to remove it. It is more practicall to avoid contaminations completely.


  • tips should be in racks, tubes — in boxes
  • use forceps for handling tubes and tips
  • place caps upside down only
  • try to organize work in such a way, that hand moves from sample to trash in one direction without turning back
  • never touch:
» internal area of tube cap. Change tube if it happens
» area neaby with the bottle neck. Protect it with aluminium foil or Saran wrap
» ends of water tubes (distilled water, water purification system). Hold tubes in ~10cm from the end.
  • hands should never come above:
» open tubes, bottles
» removed caps
» open racks with tubes and tips



[править] Dirty materials

Dirty materialPrecautions
tips, tubes, bottles, columns, etc., that were in contact with:
» eucaryotes, bacteria
» oligos, DNA, RNA: natural and cloned
» haotropic salts, detergents
» enzymes
» etc.
  • wash or throw away immediately
common equipment:
» photo-documentation system, hand-held UV-lamp
» NA analysers: spectrophotometer, NanoDrop, capillary electrophoresis, etc.,
» centrifuge, PCR machine, RT-PCR
» electrophoresis and gel-cutting table
  • wash equipment before use
  • change/wash gloves after use
common places:
» door handle
» water tap
» telephone
» light switches
» doors of fridges, freezers
» textile towel
  • do not use gloves outside of the working table
  • if use gloves outside of the working table — change/wash them
  • do not use fingers for turning on/off light, opening doors, etc.
  • use paper towel in the lab
personal things:
» laboratory journal, pen, pencil, tape
» keyboard/mouse, calculator
» tube boxes, racks,
» hair, nose, ears
  • wash gloves after sneezing, touching hair, nose, etc.
  • keep personal things clean, wipe with wet paper towel regularly:
» pipette handles, tip racks, tube boxes, forceps
» lab. journal, pens, pencils, scissors, tape
  • special precautions before sensitive/important experiments
» finish all paper/computer work before experiment
» print new paper with a protocol
» wash table, wash/change gloves
  • special precautions when visit post-PCR area
» better do not bring lab. journal into post-PCR area. Use separate papers, stick them into lab. journal lately
» do not take photos from gel-doc system with gloves
  • dirty materials should never contact clean surfaces:
» table, journal, keyboard, mouse
» racks for tips and tubes, boxes with tubes
» gloves, forceps
» pipettes
» etc.

if this happens (or you suppose, that it happened), wash surface immediately.

[править] Gloves

NB! Gloves is not a "magic bullet":
» sweat from taken out gloves have the same RNase activity, as a sweat from hands
» wash gloves if you sneeze into them
  • there are two types of gloves: with and w/o talk. Only talk free (dust free) gloves should be used in the laboratory
  • wash gloves on the hands regularly (blot with paper towel)
  • when it is too hot, put a piece of paper towel under the glove to prevent sweat dripping
  • how to take gloves out:
» do not put on clean surface turned inside out gloves
» be careful not to sputter sweat from inside of gloves
  • gloves should be worn:
» within the freezer
» RNA work
» RT PCR
» library construction
» work with common equipment (see previous paragraph)
  • change/wash gloves after contact with:
» dirty material
» common equipment
» coming from post-PCR to pre-PCR area
» before start of important or sensitive experiment



[править] How not to mix up samples

  • put tubes always in the same order: in racks, on the table, in fridge/freezer, in centrifuge, etc.
  • parallel work with several samples accelerates the process. But if the number of samples is too big, it is more practical to split them on several groups and process groups one after another
  • load samples on the gel always from left to right
  • immediately after PAAGE electrophoresis cut top-right corner of the gel
  • immediately after blot cut top-right corner of the membrane

[править] Signing

  • dispense samples only in signed tubes
  • when possible, sign tubes and dispense known components before starting a work
  • use clear distinguishable names:
1,2,3,4,5,6,7,8,9,...
A,B,C,D,E,F,...

In this respect names "a","b","c","d" is a bad choice, because "a" and "d" looks similar. Sometimes, it is more convenient to give a temporal names, than to use original ones (they may be too long, or too similar, etc.). Dangerous pairs:

"a" / "d"
"h" / "n"
"q" / "g"
"e" / "l"
"r" / "v"
"f" / "t"
"D" / "O"
"C" / "G"
  • sign both tube and cap if:
» cap is removable
» you work with organic solvents (ethanol, isopropanol, chloroform, etc.)
  • when signing caps:
» cap-tails on the top
» symmetrical cups: dot after symmetrical numbers "6 / 9", "18 / 81", etc.
  • when signing tube for storage: date is obligatory

[править] Labels, markers

  • labels:
» only Cryo-Tag labels[1] — other labels slip down in liquid N2
» for caps: Laser Tough-Spots[2]
  • only two types of markers are stable enough:
» Kryomarker, schwarz, 0.75mm [3]: thin and stable
» : bright, thick
  • marker writing on plastic difuse with time if it is covered by transparent tape



[править] How not to loose a sample

[править] NA purification

  • spin columns:
» keep "loading solution flow through" until you are sure, that sample is not lost
» do not touch silica membrane with a tip
  • Isopropanol/EtOH precipitation:
» keep "supernatant combined with 70% EtOH washes" until you are sure, that sample is not lost
» do not touch with a tip pellet-side internal part of the tube
» try to control position of the visible pellet
  • Phenol/chloroform purification of valuable samples: phase lock or additional extraction of interphase

[править] Sample solubilization

  • do not use pipetting[4]
  • give enough time, controll completness visually

[править] Tubes handling

  • spin down before opening
  • always put tubes into centrifuge in the same orientation
  • blot cap after water bath
  • use cap locks for:
» boiling
» rotation

[править] Experiment planning

  • whenever possible, create backup(s) to be able to start not from the very beginning in case of an accident:
» cut reserve gel blocks for library construction
» use part of the library (1/3-1/2) for library amplification
» plan analytical tests for couple of analysis (gel loadings)

[править] Storage

  • tube signing: date is obligatory
  • should be a system in distribution of tubes in freezers/boxes
  • special "museum" collections of valuable strains, constructions, libraries, etc.



[править] Bench

  • try to keep bench as empty as possible when it is not in use. Leave on the bench only vortex, centrifuge(s) and pipettes



[править] Centrifugation

  • wash regularly internal part of the centrifuge:
» weekly, remove rotor for washing
» after first PE/RPE wash
» before enzymes/buffers aliquoting
  • always put tubes into centrifuge in the same orientation (except of backet-rotors):
» cap-tails oriented outside
» tubes without cups: write dot by marker, orient it outside
  • big centrifuge: use smooth breaking for pelleting and phase separation (phenol and chloroform extraction, salt/glycerol gradients)
  • to accelerate rotor cooling, run centrifuge at low speed
  • good practice for removal of supernatant (EtOH precipitation, 70% EtOH wash):
▫ remove main part of supernatant (pour off, pipette, etc.)
▫ close tube and centrifuge again for 2-5min
▫ remove the rest of supernatant by aspiration
  • for precipitation of short Nucleic Acids (NA) or low amounts of NA:
» centrifuge at +4°C
» centrifuge for a long time: 15-30min



[править] Incubation at certain temperature

  • "preheating/precooling" is not required if there is a good contact with a tube:
» water bath (automatic, thermos, in foam plastic box)
» ice bath (ice with water)
» PCR machine
» liquid N2
» dry ice with ethanol
» thermoshaker with tube-form wells
  • requires special precautions (preheating/precooling):
» ice w/o water (dry ice w/o ethanol): small area of ice/tube contact
» air thermostat, thermostated room: low speed of heating. Preheat sample, put it on preheated surface or under a fan
» refrigerator, freezer: low speed of cooling. Precool (snap freeze) sample. Put sample on cold surface
  • preheating of tubes/bottles before incubation in air thermostat
▫ preheat in water bath
▫ blot water with paper towel
▫ put in air thermostat


[править] Typical problems

  • extremely low speed of heating:
» stacks of Petri dishes / bacterial plates in air thermostat
» plastic foam racks are good thermoisolators both for air thermostat and for water bath
» tube in cardboard (foam plastic) box in freezer
» cooling/heating of large volumes: small surface/volume ratio. Mix bottle from time to time
  • water is nice thermoisolator. It heat up and cool down fast only because of convection:
» cooling should be from the top, heating — from the bottom
» be sure, that bottle is completely submerged in the cooling substance: ice, dry ice, ice bath, etc.
» viscous solutions require longer time for cooling/heating
  • water bath
» water gets between tube and cap: blot, remove by centrifugation before opening
» level of sample in the tube/bottle should not be above the level of the water in the water bath
  • bucket with ice (dry ice):
» tubes and bottles should not be above the walls of the bucket
» liquid level should be below of ice level



[править] Freezer

  • use gloves; on -70°C — also forceps
  • if it is necessary to find a tube in the box never put box on the room-temperature surface (bench, top of the freezer, knee, etc.) !!!
» quick work (1-2min.): put box cover on the bench (flat surface top), put box on the cover
» long work (>2min.):
-20°C: put -20°C precooled block in the plastic foam box. Put storage box on the precooled block.
-70°C: cover bottom of plastic foam box with dry ice. Put storage box on dry ice.
Check, that box is below than plastic foam box walls. Put plastic foam cover on the top when not in use. Use forceps to handle the tubes.
  • freeze/melt samples fast[5]
freezing:
» everything except of enzymes should be snap freezed in liquid N2 before storage
» do not put "just from N2 tubes" nearby with enzymes in the freezer
melting:
» enzymes from -70°C: melt in hands until a small piece of ice remain in the tube, then put in Stratagene cool box
» competent cells: in hands until a small piece of ice remain in the tube, then put in ice
» buffer melting: in room-temperature water bath or hands until a small piece of ice remain in the tube/bottle, then put in ice
  • control freezer temperature: should be -18-25°C[6]. Some enzymes are extremely sensitive to overcooling (RNA ligase)
  • blot cold boxes with paper towel before putting them back on -20°C



[править] Automatic pipettes

  • pipettes are calibrated at room temperature, in vertical position, for water. In all other cases special precautions are necessary
  • store vertically
  • transport on tray covered by paper towel (never put into pocket!)
  • if liquid comes inside the pipette: open it and clean immediately
  • DO NOT:
» incline for more than 60° with a liquid in the tip (better, never incline for >60°)
» put on the table with a liquid in the tip (better, never put on the table at all)
» use pipette for strong acids
» let anybody use your pipettes


[править] Maintenance

  • regularly (weekly, or after suspicion on contact with dirty material) wipe pipette with paper towel:
▫ slightly damped with water
▫ slightly damped with 70% EtOH
▫ dry
  • check each month for leakage (gloves, filtered tip):
▫ aspirate ~1/2 of maximum volume of water
▫ close the end of the tip with gloved finger
▫ maximally push the pipette piston and wait for 20 sec.
▫ release the pipette piston
▫ open the end of the tip

if there is a leakage, water would rise up in the tip


  1. Sigma, #Z376914-1ROL
  2. diam. 3/8 in., white, Sigma, #T1316
  3. A.Hartenstein GmBH #MCRS
  4. pellet may stick to the internal surface of the tip
  5. freeze border can move back/forth if freezing/melting is slow
  6. Digital-Thermometer VWR Cat#620-0919, ~14€
Источник — «http://molbiol.ru/wiki/Wet_laboratory»


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