Haynes Lab:Notebook/David Barclay Undergrad Training/Plasmid Transformation/Entry Base: Difference between revisions
(7 intermediate revisions by the same user not shown) | |||
Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
<!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | ||
==Tuesday, June 25th== | |||
==== Steps for Transformation ==== | |||
---- | |||
Using KAH06 for Plasmid.<br> | |||
Take 1 μL of KAH06.<br> | |||
Add 9 μL of ddH20. <br> | |||
---- | |||
=====Notes===== | |||
---- | |||
*KAH06 has a bp length of 1170. The gene codes for hCBX8, a human chromobox protein (8). | |||
==Friday, June 28th== | ==Friday, June 28th== | ||
====Miniprep==== | |||
---- | |||
Aims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration. | |||
Protocol: <br> | |||
Using Qiaga Protocol,shown below. <br> | |||
[[Image:Lab01.jpg|200px|]] <br> | |||
Miniprep followed exactly <br> | |||
=====A. Pre-Miniprep===== | |||
1. Add 3 mL LB into 15 mL tube <br> | |||
2. Add one colony of KAH06 into 3mL LB Broth: Incubate 7 hrs at 37 C. | |||
=====B. Miniprep===== | |||
1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. <br> | |||
NOTE: REMEMBER TO MARK TUBES <br> | |||
2. Take 600 μL of sample. Add 100 μL 7x lysis buffer (blue) and invert 4 times. Time limit 2 mins for lysis. <br> | |||
3. Add 350 μL Neutralization Buffer (yellow) invert until sample become yellow (10 times). Neutralization Buffer must be refrigerated. <br> | |||
4. Centrifuge 2 mins @ 13.1g. <br> | |||
5. Transfer ~850 μL into spin column. Put spin column into collection tube. Centrifuge 15 secs @ 13.1g. Discard flow through. <br> | |||
7. Add 200 μL of elution buffer (ENDO Wash Buffer). Centrifuge 15 secs @ 13.1g. <br> | |||
8. Add 400 μL of Zyppy wash buffer into column. Centrifuge 30 secs @ 13.1g. <br> | |||
9. Transfer column into new 1.5 mL tube. Add 30 μL of Zyppy Buffer and let sit for 1 min. Centrifuge 15 secs @ 13.1 g. <br> | |||
=====C. DNA Concentration- Take 3 Plate===== | |||
1. Open program Gen 5 2.0 before machine. <br> | |||
2. Add 2 μL of sample into holes. <br> | |||
3. Use either water or buffer as blank depending on buffer DNA was added to. <br> | |||
4. Open program "Nucleic Acid Quantification" <br> | |||
5. Use DI water and kim wipe to clean plate. <br> | |||
---- | |||
====Results==== | |||
---- | |||
*1.17 μg/μL. Need between 20-80 μg/μL. <br> | |||
*0.5 260/280. Need about 1.8 260/280. Shows major contamination. <br> | |||
---- | |||
====Causes of Errors==== | |||
---- | |||
*Not exactly 600 μL of initial LB+sample. +-50 μL. '''[[Step A.2]]''' <br> | |||
*Pipette did not grab exactly 2 μL to add to Take 3 Plate. '''Step B.2''' <br> | |||
*Buffer used was Zyppy, not Water. Take 3 Plate could not have compensated. ''' Step B.3''' <br> | |||
*High contamination from disturbed pellet? '''Step B.5''' <br> | |||
*Other Contamination in Parts A, B and C. <br> | |||
Revision as of 12:56, 30 June 2013
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page |
Tuesday, June 25thSteps for TransformationUsing KAH06 for Plasmid. Notes
Friday, June 28thMiniprepAims: Run plasmids through miniprep in order to isolate plasmid DNA and determine concentration. Protocol: A. Pre-Miniprep1. Add 3 mL LB into 15 mL tube B. Miniprep1. Pipette 1.5 mL of sample into 2 mL tube. Spin for 3 min @ max setting (13.1g). Repeat once more to spin entire sample. C. DNA Concentration- Take 3 Plate1. Open program Gen 5 2.0 before machine. Results
Causes of Errors
|