Revision as of 02:50, 23 September 2013

MINIPREP

Two 100 ml culture of EColi DH5alfa carrying a plasmid with a mRFP generator under arsenite sensitive promoter ([http://parts.igem.org/Part:BBa_K1106003 Bba_K1106003]) where grown at 30°C until they reach OD=0.4 (600nm). At this point arsenite was added (10ppb and 1000ppb of arsenite final concentration in each other) fluorescence was measured every 30 minutes for 8 hours with a fluorimeter at 584 nm excitation peak and 607 nm emission peak. All these data was normalized by the culture density measured by OD.

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 = MINIPREP =
-==Materials==
+Two 100 ml culture of EColi DH5alfa carrying a plasmid with a mRFP generator under arsenite sensitive promoter ([http://parts.igem.org/Part:BBa_K1106003 Bba_K1106003]) where grown at 30°C until they reach OD=0.4 (600nm). At this point arsenite was added (10ppb and 1000ppb of arsenite final concentration in each other) fluorescence was measured every 30 minutes for 8 hours with a fluorimeter at 584 nm excitation peak and 607 nm emission peak. All these data was normalized by the culture density measured by OD.
-* Resuspension buffer
-* Lysis buffer
-* Neutralization buffer
-* Isopropanol
-* 70% Etanol
-* dH2O
-* Ice (in ice bucket/container)
-==Procedure==
-# Centrifuge for 5 minutes at 5000 rpm 1,5 ml of bacteria culture.
-# Take the pellet and add 250 ul of resuspension buffer and pipet up and down a few times.(make sure that all is properly mixed)
-# Add 250 ul of Lysis buffer and invert the tubes 5 times to mix. Let rest 5 minutes. The mix must turn transparent.
-# Add 250 ul of cold Neutralization buffer and invert the tubes 5 times to mix. The mix must turn turbid.
-# Centrifuge at 13.000 rpm for 15 minutes.
-# Transfer the supernatant to a new clean eppendorf tube.
-# Add isopropanol (the same volume that is in the tube). Mix many times and put it in ice for 20 minutes.
-# Centrifuge at 13.000 rpm for 20 minutes.
-# Eliminate the supernatant (be carefull that the pellet does not go with it)
-# Add 1 ml of etanol 70% and use a vortex to ensure the pellet mixes with the etanol.
-# Centrifuge at 13.000 rpm for 5 minutes.
-# Eliminate all the supernatant with a pipette (be carefull that the pellet does not go with it)
-# Let the tube open until it is dried. The pellet must turn from white to transparent.
-# Add 20 ul of dH2O and freeze.
-=Transformation=
-We have E. Coli DH5α strain competent bacteria, with a transformation efficiency of 10
-'''Important!''' Competent bacteria should always be on ice.
-# If the plasmid comes from the kit or is a product of ligation, add 2μl of plasmidic DNA into 50μl
-of competent bacteria. If it’s a plasmid that comes from a miniprep, use a mass in function of
-the efficiency of the bacteria.
-# Incubate for 20 min on ice.
-# Heat Shock: 42°C for exactly 1’30’’, then immediately place on ice.
-# Incubate for 5 min on ice.
-# Recovery: Add 950μl of LB without antibiotic, incubate from 30 min to an hour at 37°C
-# Pellet the bacteria by centrigue at 10000rpm for 5 minutes. Discard the supernatant of LB.
-Resuspend the bacteria in the remnant LB.
-# Plaque onto plates with LB and the desired antibiotic.
-# Incubate overnight in a stove at 37°C
-=Ligation=
-'''Important!''' Before starting the ligation process, make sure that the restriction enzymes are inactive.
-# Add 2μl of digested plasmid backbone (or the equivalent of 25ng).
-# Add equimolar amount of EcoR1-HF Spe1 digested fragment (less than 3μl).
-# Add equimolar amount of Xba1 Pst1 digested fragment (less than 3μl).
-# Add 2μl of T4 DNA ligase buffer (final concentration should be 1x).
-# Add 0.5μl of T4 DNA ligase.
-# Add water to 10μl.
-# Ligate at 16°C for 30 min, then heat kill 80°C for 20min.
-# Transform with 1 – 2 μl of product.
-=LB & LB agar=
-# Liquid LB (1 litre)
-# 10g of bacto peptone (peptone, triptone or bactereologic triptone)
-# 5g of yeast extract
-# 10g of NaCl
-# Fill with dH2O
-Autoclave.
-TIP: if it is not going to be autoclaved in the next few hours water should not be added (because even
-when in the fridge it will get contaminated), in which case everything can be measured and left ready to
-just add the water.
-# LB agar (1 litre)
-# 10g of bacto peptone (peptone, triptone or bactereologic triptone)
-# 5g of yeast extract
-# 10g of NaCl
-# 15g of agar
-# Fill with dH2O
-'''IMPORTANT''': agar does not dissolve if not heated so if Erlen-Meyers are prepared with LB agar, add LB in
-liquid form and then add the agar to the Erlen-Meyer according to the volume.
-Autoclave.
-'''TIP''': if it is not going to be autoclaved in the next few hours water should not be added (because even
-when in the fridge it will get contaminated), in which case everything can be measured and left ready to
-just add the water.
-=Gel=
-TAE 1x (diludde from TAE stock solution – 50x)
-Agarose
-Remember to use gloves during the entire process, being mindful not to have direct contact with
-ethidium bromide.
-# Add the necessary agarose into an Erlenmeyer flask and then add TAE, and fill with water. The
-final concentration of agarose may depend on the type of gel needed, but usually will be 1%.
-#  Heat the flask, while covered,  in a microwave until completely dissolved (should take no more
-than 3 minutes at maximum potency)
-#  Add the necessary volume of Ethidium Bromide (about 10 μl for each 100g of gel)
-'''IMPORTANT''': Ethidium Bromide is carcinogenic. It should be handled with gloves, and these,
-such as anything that has come in direct contact with Ethidium Bromide must be discarded
-separately in an assigned discard bag.
-# Pour the melted gel, with Ethidium Bromide, slowly into the gel tray. Add the combs and leave
-until solified. Remove the combs. Rinse the flask with abundant water.
-#  Place the gel tray inside the gel box being mindful of the direction in which gel will run. Add TAE
-x until it is completely covered.
 </div>

Team:Buenos Aires/ resqrfp

From 2013.igem.org

Revision as of 02:50, 23 September 2013

MINIPREP