IGEM:Cambridge/2008/Protocols: Difference between revisions
No edit summary |
No edit summary |
||
Line 155: | Line 155: | ||
:*Add 200μL Wash Buffer. Spin for 30 seconds. Repeat wash step. | :*Add 200μL Wash Buffer. Spin for 30 seconds. Repeat wash step. | ||
:*Place column into Eppendorf. Add 6-10μL SDW to elute DNA. Add more SDW to elute more DNA - normally 20μL. | :*Place column into Eppendorf. Add 6-10μL SDW to elute DNA. Add more SDW to elute more DNA - normally 20μL. | ||
=Glycerol Stock (Modified Version)= | |||
:*Inoculate single colony growing on plate into 10 ml LB with appropriate selection. Grow overnight. | |||
:*Add 100μL culture to 500μL sterile 80% glycerol stock (need to sterilize using syringe). | |||
:*Vortex briefly | |||
:*Freeze at -80 degrees. | |||
=Media Preparation= | |||
==NOK 2.0 (No potassium nutrient broth)== | |||
Measure out: | |||
:*6.0g Na2HPO4 | |||
:*3.0g NaH2PO4 | |||
:*0.5g NaCl | |||
:*1.0g NH4Cl | |||
:*0.0147g CaCl2 | |||
:*0.246g MgSO4.7H2O | |||
:*2.0g Glucose | |||
:*1.0g NOK Amino Acid Powder (see below) | |||
Add to 1dm3 (1l) of SDW and shake well. Note heating 40-50°C may be necessary to achieve full dissolution of solutes. There may be a small amount of indeterminate crud remaining at the end of this process; this is entirely normal and can be ignored. This mixture should then be filter sterilised into 50ml separate tubes (reduces contamination risk of whole batch). Keep refrigerated or frozen until needed. | |||
'''NOK Amino Acid Powder composition''' | |||
This is a solid homogenized mixture of all solid amino acids (except possible GluR0 channel agonists alanine, threonine, serine, glycine, glutamine & glutamate.) | |||
Current batch is stored at room temperature on the chemical shelf near the gel area. To make a new batch, measure out 0.5g of each of the following (L-forms ONLY): | |||
:*Arginine | |||
:*Asparagine | |||
:*Aspartic Acid | |||
:*Cystein | |||
:*Histidine | |||
:*Isoleucine | |||
:*Leucine | |||
:*Lysine | |||
:*Methionine | |||
:*Phenylalanine | |||
:*Proline | |||
:*Tryptophan | |||
:*Tyrosine | |||
:*Valine | |||
=Oxygen Electrode= | =Oxygen Electrode= | ||
Line 170: | Line 210: | ||
:*Add 400μL Zyppy Wash. Spin for 30 seconds. Repeat this step one more time for better purity. | :*Add 400μL Zyppy Wash. Spin for 30 seconds. Repeat this step one more time for better purity. | ||
:*Transfer column to clean Eppendorf. Add 30 to 100μL Elution Buffer to the column and spin for 15 seconds to elute DNA | :*Transfer column to clean Eppendorf. Add 30 to 100μL Elution Buffer to the column and spin for 15 seconds to elute DNA | ||
=Primer Preparation and Storage= | =Primer Preparation and Storage= | ||
Line 212: | Line 246: | ||
After transformation cells need to be grown under selection conditions. | After transformation cells need to be grown under selection conditions. | ||
Revision as of 07:40, 3 September 2008
Agarose GelUsed for Electrophoresis when extracting any product afterwards. Making the Gel
Loading the Gel
Running the Gel
Visualising the Gel
Biobrick ExtractionWe found we could not extract enough DNA from the registry, so we are upgrading the extraction to the "bigger, better, faster, stronger" method.
We then confirmed with PCR. Competent Cells StocksChemically competent cells are made by growing overnight in 200ml LB in a shaking incubator at 37°C. 200ml are spun down at 3800rpm for 8 mins, resuspended in 20ml CaCl2, spun down again and resuspended in 4ml CaCl2. Again spun down (6500 rpm for 5 mins)and resuspended in 4ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they are aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. To recover cells thaw the tubes on ice, spin down and resucpend in 100μL ice cold CaCl2. Electroporation competent TOP10 and DH5α cells were made by growing overnight in 200ml LB in a shaking incubater at 37°C. 100ml were spun down at 3800rpm for 8 mins, resuspended in 25ml SDW, spun down again and resuspended in 2ml SDW. Again they were spun down (6500 rpm for 5 mins)and resuspended in 2ml 60%glycerol, spun down and resuspended in 2ml 60%glycerol. Finally they were aliquoted (100μL) into stock tubes and flash frozen in liquid nitrogen. To recover cells thaw the tubes on ice, spin down and resucpend in 100μL ice cold SDW. Dephosphorylation
E-GelE-Gel is used to confirm size of products, but not for extraction.
Fast DigestionThe following are added to a 1.5μL eppendorf:
If using PCR product the whole is made up to 30μL, using 10μL DNA and 3μL buffer. The tubes are then incubated at 37°C for 5mins and then the enzyme inactivated at 80°C for 15mins Fast LigationThe following are added to a 1.5μL eppendorf:
The tubes are then incubated at room temperature for 5mins and then the enzyme inactivated at 75°C for 15mins We have now got a different fast ligation kit. Now the following are added to a 1.5μL eppendorf:
The tubes are then incubated at room temperature for 5mins. Flame PhotometrySet Up
Taking readings
In case of compressor blow-out
In case of wildly fluctuating "zero" readings
Gel DNA Recovery (Zymoclean)
Glycerol Stock (Modified Version)
Media PreparationNOK 2.0 (No potassium nutrient broth)Measure out:
Add to 1dm3 (1l) of SDW and shake well. Note heating 40-50°C may be necessary to achieve full dissolution of solutes. There may be a small amount of indeterminate crud remaining at the end of this process; this is entirely normal and can be ignored. This mixture should then be filter sterilised into 50ml separate tubes (reduces contamination risk of whole batch). Keep refrigerated or frozen until needed. NOK Amino Acid Powder composition This is a solid homogenized mixture of all solid amino acids (except possible GluR0 channel agonists alanine, threonine, serine, glycine, glutamine & glutamate.) Current batch is stored at room temperature on the chemical shelf near the gel area. To make a new batch, measure out 0.5g of each of the following (L-forms ONLY):
Oxygen ElectrodeFor the protocol see http://www.rankbrothers.co.uk/download/digioxy.pdf Plasmid Miniprep (Zyppy)
Primer Preparation and StorageSigma Primers arrived as tubes of dried oligos
PCRTo make 50μL of PCR mix the following were added to Eppendorf tubes:
These values can be adjusted for any volumes of final mixture. The total final volume is transferred to thin walled tubes (may need to be spun down) and then placed in a PCR machine and the appropriate program run. TransformationFirst chemically competent cells are made
To transform cells
After transformation cells need to be grown under selection conditions. |