Summary
Summary BMW Molecular Techniques Lab Journal
- Course
- Institution
The lab journal with notes for the course in molecular techniques.
[Show more]
Seller
Follow
charlottevandervoort
Reviews received
Content preview
LABJOURNALCharlotte van der Voort
13653318
The effect of TRP on PP5
A scientific investigation of the function of the
tetratricopeptide repeat (TPR) domain on the
phosphatase activity of the enzyme
phosphatase (PP5) in Rattus norvegicus
,Content
Gene editing…………………………………………………………………………………………………….4
Title…………………………………………………………………………………………………………………4
Goal…………………………………………………………………………………………………………………4
Predictions and/or expectations…………………………………………………………………………4
Agarose gel electroforesis………………………………………………………………………..4
Experimental design………………………………………………………………………………………….5
PCR ..……………………………………………………………………………………………….……5
Agarose gel electroforesis………………………………………………………………………..5
Log-content………………………………………………………………………………………………………6
PCR.………………………………………………………………………………………………….…..6
Agarose gel electroforesis………………………………………………………………………..7
Results……………………………………………………………………………………………………………..7
Agarose gel electroforesis ……………………………………………………………………….7
Conclusion/ discussion ……………………………………………………………………………………..8
Transformation…………………………………………………………………………………………………9
Title………………………………………………………………………………………………………………….9
Goal………………………………………………………………………………………………………………….9
Predictions and/or expectations………………………………………………………………………….9
DNA purification…………………………………………………………………………………….9
Golden Gate assembly.…………………………………………………………………………….9
Transform E.coli BL21 Gold.…………………………………………………………………..10
Agarose gel electroforesis after colony PCR....…………………………………………..10
Agarose gel electroforesis after DNA digestion..………………………………………..11
Sanger sequence………..…………………………………………………………………………..11
Experimental design………………………………………………………………………………………….11
DNA purification……………………………………………………………………………………12
Golden Gate assembly.……………………………………………………………………………12
Transform E.coli BL21 Gold.……………………………………………………………………12
Colony PCR……………………………………………………………………………………………13
Agarose gel electroforesis……………………......…………………………………..............13
Plasmid DNA isolatation…………………………………………………………………………13
Sanger sequence………..…………………………………………………………………………..14
Log-content………………………………………………………………………………………………………14
DNA purification……………………………………………………………………………………14
Measure DNA concentration after DNA purification…………………….……………14
Golden Gate assembly.……………………………………………………………………………15
Prepare agar plates…………………………………………………………………….…………..15
Transform E.coli BL21 Gold.………………………………………………………...............16
Colony PCR…………………………………………………………………………………………...16
Inspect plates and prepare cell suspension……………………………………………....18
Agarose gel electroforesis after colony PCR..……………………………………………..19
Reinoculate cells for protein expression……………………………………………………19
Plasmid DNA isolatation…………………………………………………………………………19
DNA digestion BglII/SmaI……………………………………………………………………..20
Measure DNA concentration after plasmid DNA isolation………………………….21
Agarose gel electroforesis after DNA digestion……………………………………….…21
Sanger sequence………..…………………………………………………………………………..21
2
RAPPORTTITEL
, Results………………………………………………………………………………………………………….…22
Agarose gel electroforesis after colony PCR.……………………………………………..22
Agarose gel electroforesis after DNA digestion………………………………………….23
Sanger sequence…………………………………………………………………………………….23
Conclusion/ discussion …………………………………………………………………………………….24
Protein expression……………………………………………………………………………………………26
Title………………………………………………………………………………………………………………..26
Goal………………………………………………………………………………………………………………..26
Predictions and/or expectations………………………………………………………………………..26
Induce protein expression and lyse cell…………………………………………............26
His-tag purification………………………………………………………………………………..26
Transform E.coli BL21 Gold.…………………………………………………………………...26
Mass spectrometry analysis....…………………………………………………….………..…26
SDS-PAGE ..…………………………………………………………………..……………………..26
Bradford assay ………..………………………………………………………….…….……….….26
Western blot ………..………………………………………………………………..……………...26
Coomassie staining………..…………..…………………………………………………………..27
Experimental design………………………………………………………………………………………...27
Induce protein expression and lyse cell………………….………………………………..28
SDS-PAGE ..………………………………………………………………………………………....28
Bradford assay ………..…………………………………………………………………………….28
Mass spectrometry analysis....……………………………………….………………………..29
Western blot ………..……………………………………………………………………………….29
Log-content……………………………………………………………………………………………………..29
Induce protein expression and lyse cell…………………………………….……………...29
SDS-PAGE ..………………………………………………………………………………….……...30
Prepare for Mass spectrometry…………………………………………………………………31
His-tag purification…………………………………………………………………………………31
Bradford assay ………..……………………………………………………………………………..31
SDS-PAGE ..…………………………………………………………………………………………..34
Western blot ………..………………………………………………………………………………..34
Coomassie staining………..………..……………………………………………………………..34
Mass spectrometry analysis....…………………………………….…………………………..35
Results…………………………………………………………………………………………………………….35
Coomassie staining………..……………………..…………………………………………….….35
Western blot…………………………………………………………..……………………………..36
Mass spectrometry………………………………………………………………………………...36
Conclusion/ discussion …………………………………………………………………………………..…37
Phosphatase activity…………………………………………………………………………………………38
Title………………………………………………………………………………………………………………..38
Goal………………………………………………………………………………………………………………..38
Predictions and/or expectations………………………………………………………………………..38
Phosphatase assay………………………………………………………………………………...38
Experimental design…………………………………………………………………………………………38
Phosphatase assay…………………………………………………………………………………38
Log-content……………………………………………………………………………………………………..39
Phosphatase assay…………………………………………………………………………………39
Results…………………………………………………………………………………………………………….40
Phosphatase assay………………………………………………………………………………….40
Conclusion/ discussion …………………………………………………………………………………….41
3
RAPPORTTITEL
, Title
Developing an pET28-a vector with RnPP5-FL and RnPP5-ΔTPR, using a PCR and
Agarose gel electroforesis
Goal
Amplifying the DNA with the RnPP5-FL and RnPP5-ΔTPR DNA fragments and
ligating them into a pET28-a vector, using a PCR and Agarose gel electrophoresis, to
be used in the next experiment for the transformation of an E.coli BL12 Gold.
Predictions and/or expectations
- MPP PP5 C by phosphate domain(light green)
- PLN03088 by TPR domain (light blue)
- 4JA9 A (light pink)
- Active site (yellow)
- Metal binding site (magenta)
- TPR interaction site (red)
- Putative protein binding surface (orange)
Figure 1: The domains of the PP5
protein. This figure is made with
snapgene.
Agarose gel electroforesis
- The expected length of the PP5-FL PCR product is 1528 bp, while the expected
length of the PP5-ΔTPR PCR product should be 1051 bp.
- The primers in the blank will bind together and leave a vague band on the gel
as a result.
Figure 2: The expected length of the PP5-FL PCR product is 1528 bp. This figure is made with snapgene.
Figure 3: The expected length of the PP5-ΔTPR PCR product is 1051 bp. This figure is made with snapgene.
4
RAPPORTTITEL
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller charlottevandervoort. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $6.89. You're not tied to anything after your purchase.
Can Stuvia be trusted?
4.6 stars on Google & Trustpilot (+1000 reviews)
48072 documents were sold in the last 30 days
Founded in 2010, the go-to place to buy study notes for 15 years now