Difference between revisions of "20.109(F16):Induce CRISPRi system (Day7)"
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'''Retrieve sequence results from Genewiz''' | '''Retrieve sequence results from Genewiz''' | ||
#Use the pgRNA_target Benchling file that you generated on [[20.109(F16):Confirm_gRNA_sequence_(Day5)#Part_3:_Prepare_pgRNA_clones_for_sequencing_analysis| M2D5]] to confirm that the gRNA sequence you designed was indeed inserted into the vector. | #Use the pgRNA_target Benchling file that you generated on [[20.109(F16):Confirm_gRNA_sequence_(Day5)#Part_3:_Prepare_pgRNA_clones_for_sequencing_analysis| M2D5]] to confirm that the gRNA sequence you designed was indeed inserted into the vector. | ||
− | #Your sequencing data | + | #Your sequencing data is available from [http://genewiz.com this link Genewiz]. |
#*Choose the "Login" link and then use "nllyell@mit.edu" and "be20109" to access your results. | #*Choose the "Login" link and then use "nllyell@mit.edu" and "be20109" to access your results. | ||
#*At the bottom right should be a link to download your sequencing results. | #*At the bottom right should be a link to download your sequencing results. |
Revision as of 17:50, 11 October 2016
Contents
Introduction
dual plasmid system...
aTc induction...
Protocols
Part 1: Communication Lab workshop
Our communication instructors, Dr. Sean Clarke and Dr. Diana Chien, will join us today for a workshop on organizing and structuring your Research article.
Part 2: Examine pgRNA sequencing results
Your goal today is to analyze the sequencing data for you two potential mutant pgRNA clones - two independent colonies from your amplification reaction - and then decide which colony to proceed with for the CRISPRi manipulation of the E. coli MG1655 fermentation pathway.
Retrieve sequence results from Genewiz
- Use the pgRNA_target Benchling file that you generated on M2D5 to confirm that the gRNA sequence you designed was indeed inserted into the vector.
- Your sequencing data is available from this link Genewiz.
- Choose the "Login" link and then use "nllyell@mit.edu" and "be20109" to access your results.
- At the bottom right should be a link to download your sequencing results.
- TR section: click on the Tracking Number XXX (Order Date XX/XX/2016).
- WF section: click on the Tracking Number XXX (Order Date XX/XX/2016).
- The quickest way to start working with your data is to follow the "View" link under the Seq File heading. For ambiguous data, you may want to look directly at the Trace File as well.
You should align your sequencing data with a known sequence, in this case the gRNA target sequence you selected, to identify any unintended base changes that may have occurred. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using Benchling are below. Please feel free to use any program with which you are familiar.
Align with Benchling
If both colonies for your mutant have the correct sequence, choose one to use for the co-transformation step. If only one is correct, then this is the one you will use next time. If neither of your plasmids carry the appropriate gRNA target sequence, talk to the teaching faculty.
Part 3: Prepare media for mixed-acid fermentation inoculations
Reagents
Next day: | Measure fermentation products
Previous day: Journal club II