Vector NTI exercises
September 14th and 15th
Bioinformatics exercises in MBV4010 - 2015
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Vector NTI exercises
September 14th and 15th
Bioinformatics exercises in MBV4010 - 2015
page2
(Explorer MODULE. Make your notes here to remember the most important BUTTONS and their functions)
(Vector NTI MODULE. Make your notes here to remember the most important BUTTONS and their functions)
Vector NTI exercises
September 14th and 15th
Bioinformatics exercises in MBV4010 - 2015
page3
Introduction
In this lab we will perform the cloning experiments and analyses you did in the wet lab in silico,
which is using bioinformatics. We will use the software Vector NTI, a commercial tool for
sequence manipulation. This software will be new to most of you and the lab is therefore based
on the "learning by doing" principle.
You will find some numbered tips and guides marked with ( ) that refer to the “Tip Collection”.
You can also consult the TUTORIAL (http://folk.uio.no/egrini/vnti/TUTORIAL) or ask the
teachers.
Materials
An electronic copy of these exercises can be found in Fronter and links to supporting files found
at http://folk.uio.no/egrini/vnti
Assignment
You will electronically submit a Word file containing the items indicated in the exercises (maps
of molecules, digests, cloning results and answer to questions) and answers to the questions
presented at each task. You should upload the file to the appropriate folder (VectorNTI Assignment) in Fronter.
The deadline for submitting the assignment has been set to 23.59 on Sunday the 20th of
September. The idea is that this should not involve substantial homework and you should be able
to answer the questions sufficiently during the time in the computer lab.
Remember to save your work continuously! Save/screen-shot your VNTI figures
and maps to use for your report. Include a short description/figure text for the
different figures in your report.
Vector NTI exercises
Bioinformatics exercises in MBV4010 - 2015
September 14th and 15th
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Exercises
I. Cloning of the plasmids pET-28-hABH1 and pET-28-AlkB.
1. Retrieve the DNA sequences encoding hABH1 (NM_006020.2) and E. coli AlkB (J02607.1)
from NCBI (http://www.ncbi.nlm.nih.gov and make them VectorNTI entries. (See Tip1 for a
suggestion how to do this!)
2. Retrieve the DNA sequence of pET-28a (+) from http://www.merckmillipore.se and make it a
VectorNTI entry. (See Tip2 for a suggestion how to do this!)
3. By combining the retrieved sequences and information found in your course book (Section 3.2
and 4.2 of “Cloning and Mutagenesis” part), use Vector NTI to construct the sequence of the
plasmids pET-28-hABH1 and pET-28-AlkB. (See Tip3). Since you are using restriction
digestion and ligation, the restriction sites will be retained in the new constructs of pET-28hABH1 and pET-28-AlkB. (Figures for report)
4. Generate maps of the plasmids, indicating the cleavage sites (if any) for the restriction enzymes
BamHI, EcoRI, EcoRV, HindIII, NcoI, NdeI, NheI, NotI, and XhoI.
Make a list of the expected fragments and make a table for report. (See Tip4 and Tutorial for
suggestions on how to do this!).
Try to run a virtual gel showing your digests (Figure for report).
(See Tutorial)
II. Cloning of the plasmids pET-28-hABH1-del(348-389) and pET-28-AlkB-L170A
5. By using information (primer sequences etc.) from your course book, generate the sequence of
the plasmids pET-28-hABH1-del(348-389) (Figure 5, section 3.2 of course book) and pET-28AlkB-L170A (Figure 6, section 4.2 of course book).
(See Tip5) For pET-28-hABH1-del(348389), the cloning strategy is to introduce new restriction sites to the hABH1 gene which enables
you to delete the end part of the gene using restriction enzymes. For pET-28-AlkB-L170A, we
use site directed mutagenesis which will introduce a mutation in your gene construct at a
particular position. This mutation can be identified by the appearance of a new restriction site.
6. Generate restriction maps of pET-28-hABH1-del(348-389) and pET-28-AlkB-L170A, using
the same enzymes as in "4.". Include the maps of the construct in your report.
7. Calculate the fragments resulting from the following restriction enzyme digests, present the
result in a table in your report:
- pET-28-hABH1 with BamHI and NotI
- pET-28-hABH1-del(348-389) with BamHI and NotI
- pET-28-hABH1 with NdeI and XhoI
- pET-28-hABH1-del(348-389) with NdeI and XhoI
- pET-28-AlkB with NheI and EcoRV
- pET-28-AlkB-L170A with NheI and EcoRV
Vector NTI exercises
September 14th and 15th
Bioinformatics exercises in MBV4010 - 2015
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III. RT-PCR
8. In the wet lab you used primers specific for one gene and subcloned the obtained RT-PCR
fragment. Identify the mRNA sequence that you used in the wet lab at NCBI and save the
sequence in GenBank format (.gb). Import the sequence to VNTI and save it in a new folder in
VNTI Explorer. Alternatively, you can also do this using the Gene ID (GI). (See Tip1).
The mRNA sequences of SUVR1, SUVR2, SUVR4, ASHR3, ASHH2 and ASHH3 are described
in the Materials and Methods section in Baumbusch et al., 2001, Thorstensen et al., 2008 and
Grini et al., 2009 (AF394239, AY045576, AF408062, AY050894, NM_106379, AF408060)
9. Make VNTI oligo (primer) entries of the RT primers you used and save to the database.
You find the RT-PCR primers used in the course at the link http://folk.uio.no/egrini/vnti/RTPCR/
(You can also download the file “RT_primers.oa4” and import it as an oligo archive)
10. Identify primer sites using “Display_Setup/Motif”. Annotate the sequence to display the
primers and the PCR-product. Save the annotated molecule with primers and use it in your report.
Include answers to these questions in your report: Is the calculated RT-PCR product the same as
found experimentally in the lab? What would the length of an RT-PCR on genomic DNA be?
(To see genomic DNA fragments you must identify the GENOMIC DNA SEQUENCE at NCBI,
or you can download the file” genomic_RTPCR.ma4” at http://folk.uio.no/egrini/vnti/RTPCR/)
Vector NTI exercises
Bioinformatics exercises in MBV4010 - 2015
September 14th and 15th
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IV. Subcloning into pCR2.1
11. Identify the plasmid pCR2.1-TOPO from www.invitrogen.com , download to VNTI Explorer
and save to database. (Alternatively you can download pCR2.1-TOPO.gb from
http://folk.uio.no/egrini/vnti/TOPO/
12. Perform a TOPO Cloning of your RT-PCR fragment into pCR2.1 and save the molecules to
your report. You get two output options. Why? When you perform this exercise you select T-A
termini. In what case would you have selected a blunt termini? (Discuss these questions in your
report.) (See Tip12 for a suggestion how to do this!)
13. Select the output option that corresponds to the result you obtained in the wet lab. Perform a
restriction digest as you did in the lab. Calculate the fragment lengths. Do you find a match with
the results you obtained in the lab? What could be the reason for a different result? Discuss this in
your report.
14. Download the TOPO clones you have sequenced in the wet-lab and make it a VNTI entry.
(http://folk.uio.no/egrini/vnti/sequencing). (See Tip14 for a suggestion how to do this!)
15. Perform a BLAST search of your sequence using the Tools menu / Blast search.
Identify the highest hit and download the hit sequence – save it to VNTI Explorer.
Drag the found hit molecule into the program Invitrogen/AlignX and assemble with your
obtained sequencing results and your RT-PCR product. Include the alignment in your report.
V. Subcellular localization: Construction of expression vectors
16. Perform the Gateway BP reaction (BR) to create the DONOR vector you used in the lab. You
will need to download the vector pDONR-Zeo from invitrogen.com or
http://folk.uio.no/egrini/vnti/GATEWAY/. (See Tip16 for a suggestion how to do this!). In your
report, include the molecule after performing the BP-reaction.
17. Perform the Gateway LR reaction (LR) into pK7WGF2. (See Tip17).
You will find the pK7WGF2 vector at http://folk.uio.no/egrini/vnti/GATEWAY/
In your report, include the molecule after performing the LR-reaction.
Is your cDNA in frame with the GFP?