IGEM:The Citadel/Training: Difference between revisions
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=May Crash Course in Synthetic Biology= | =May Crash Course in Synthetic Biology= | ||
In order to get some serious training under our belt, do something that ''helps iGEM, and start work that can be credited towards our project'' we will be testing Biobrick promoter strength on May 28-June 4th. | In order to get some serious training under our belt, do something that ''helps iGEM, and start work that can be credited towards our project'' we will be testing Biobrick promoter strength on May 28-June 4th. | ||
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* Note that we WILL NOT be doing Step #1 (synthesis and annealing of oligos to make a test promoter). Instead, we will be testing promoters that have been included with our distribution. | * Note that we WILL NOT be doing Step #1 (synthesis and annealing of oligos to make a test promoter). Instead, we will be testing promoters that have been included with our distribution. | ||
=COMPLETE PROTOCOL= | =COMPLETE PROTOCOL= | ||
==[[Knight:Preparing_electrocompetent_cells| TK's PREPARING ELECTROCOMPETENT CELLS]]== | |||
==[[ | |||
===Materials=== | ===Materials=== | ||
<u>Equipment</u> | |||
*-80°C freezer | *-80°C freezer | ||
*37°C incubator | *37°C incubator | ||
*Refrigerated centrifuge that accepts 225 mL culture tubes | *Refrigerated centrifuge that accepts 225 mL culture tubes | ||
<u>Chemicals and Reagents</u> | |||
*~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | *~500 mL LB Lennox supplemented with appropriate concentration of antibiotic if appropriate. | ||
*~600 mL sterile deionized water chilled to 4°C | *~600 mL sterile deionized water chilled to 4°C | ||
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*Ice bucket and ice | *Ice bucket and ice | ||
*Dry ice, ethanol bath | *Dry ice, ethanol bath | ||
<u>Supplies</u> | |||
*Many 1.5 mL plastic tubes chilled to -80 °C | *Many 1.5 mL plastic tubes chilled to -80 °C | ||
*14 mL culture tube for starter culture | *14 mL culture tube for starter culture | ||
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If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. | If you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. | ||
====[[Miniprep/Qiagen_kit| MINIPREP (BASED ON THE QIAGEN KIT)]]==== | |||
== | |||
===Materials=== | ===Materials=== | ||
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*Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields. | *Heating the elution buffer to 55°C prior to loading on the column can slightly increase yields. | ||
*Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields. | *Similarly, doing the elution in two steps (first a 30 μL elution and then a 20 μL dilution) can also slightly increase yields. | ||
==[[Qiagen_Buffers| QIAGEN BUFFERS AND COLUMN RECYCLING]]== | ==[[Qiagen_Buffers| QIAGEN BUFFERS AND COLUMN RECYCLING]]== | ||
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Clicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. | Clicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. | ||
==NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUAL== | ==[http://ginkgobioworks.com/support/ NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUAL]== | ||
The above link redirects you to the protocol we will use for 3A standard assembly. | |||
==TK's RESTRICTION DIGEST== | ==[[Knight:Restriction_Digest| TK's RESTRICTION DIGEST]]== | ||
<u>Materials</u> | <u>Materials</u> | ||
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* 4°C forever (or until you pull the reaction out of the thermal cycler). | * 4°C forever (or until you pull the reaction out of the thermal cycler). | ||
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. | 7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. | ||
==Also Worth Checking Out== | |||
[[Endy:Making_a_long_term_stock_of_bacteria| DREW ENDY'S PROTOCOL FOR PRODUCING A STOCK OF BACTERIA]] | |||
[[TOP10_chemically_competent_cells| OWW's TOP10 CHEMICALLY COMPETENT CELLS]] |
Latest revision as of 15:25, 28 May 2010
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May Crash Course in Synthetic BiologyIn order to get some serious training under our belt, do something that helps iGEM, and start work that can be credited towards our project we will be testing Biobrick promoter strength on May 28-June 4th.
COMPLETE PROTOCOLTK's PREPARING ELECTROCOMPETENT CELLSMaterialsEquipment
Chemicals and Reagents
Supplies
Procedure
Notes
This protocol is for transforming plasmid DNA into Escherichia coli cells. Materials
For the following, you need one per DNA sample
Procedure
NotesIf you are in a hurry and your selection marker is ampicillin, you can go ahead and plate immediately because ampicillin takes a while to be pumped into cells at a high enough concentration to have an effect. MINIPREP (BASED ON THE QIAGEN KIT)MaterialsFor purifying plasmid DNA from Escherichia coli cells, the Qiagen Spin Miniprep Kit produces quite reliable results.
Buffer P1
The buffer and RNaseA can also be ordered from Qiagen separately (catalog numbers 19051 and 19101). Buffer P2
Buffer P3 (not for spin columns, but for Qiatips, midi, maxi, giga kits)
Buffer N3
Buffer PB
Buffer PE
Buffer QBT equilibration buffer
Buffer QC wash buffer
Buffer QF elution buffer
Buffer QN
The LyseBlue indicator dye added to some of the buffers is Thymophthalein, pH shift from colorless to blue at pH 9.3 ProtocolSee here or here for the handbook for the Qiagen Spin Miniprep Kit. If you have never done this protocol before, read the the background information in the handbook (like the Important Notes section). It contains useful information. The following has been reproduced from the handbook and annotated based on experience with the kit. Protocol: QIAprep Spin Miniprep Kit Using a Microcentrifuge This protocol is designed for purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, refer to the recommendations on page 37. Please read “Important Notes” on pages 19–21 before starting. Note: All protocol steps should be carried out at room temperature. Procedure
Notes
QIAGEN BUFFERS AND COLUMN RECYCLINGClicking the link above will provide you with the compositions of the different buffers used by Qiagen, as well as instructions on how to conserve the column that are included with the kit. NEB/GINKGO'S 3A BIOBRICK ASSEMBLY MANUALThe above link redirects you to the protocol we will use for 3A standard assembly. TK's RESTRICTION DIGESTMaterials
Digest Mix Example - 50 μL reaction. 100 μL reactions are also common especially if your DNA to be cut is dilute.
Procedure
3. Add BSA to the tube.
4. Add appropriate amount of DNA to be cut to the tube.
5. Add 1 μL of each enzyme.
6. Place in thermal cycler (MJ Research, PT-200) and run digest protocol.
7. Generally, use some method of DNA purification to eliminate enzymes and salt from the reaction. Also Worth Checking Out |