Difference between revisions of "Part:BBa K1352006:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
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Restriction digest + Gel electrophoresis
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Firgure 1; a Xba1 + HindIII restriction digest screen of recombinants.  The recombinant which went on to become K1352006 is in lane 5.  The “L” lane is DNA marker (ladder).  The arrows are to highlight the distance travelled by the HindIII-negative recombinants.
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Figure 2; a restriction digest verification of plasmid K1352006
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For figure 2: the letters indicate the following:
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L. 10,000bp – 500bp DNA marker “ladder”
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N. K1352006 plasmid digested with no enzymes
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E. K1352006 plasmid digested with EcoRI
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X. K1352006 plasmid digested with XbaI
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S. K1352006 plasmid digested with SpeI
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P. K1352006 plasmid digested with PstI
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EP. K1352006 plasmid digested with EcoRI and PstI
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XS. K1352006 plasmid digested with XbaI and Spe1
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DNA Sequencing
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The recombinant plasmid was Sanger-sequenced with the following sequencing primers.  “G101” is a reverse primer, the rest are forward primers.
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“G101” (attaccgcctttgagtgagc)
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“G100” (tgccacctgacgtctaagaa)
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“35 INP-SEQ 1” (ccgattcattaatgcagctgg)
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“36 INP-SEQ 2” (gaggttgctgttgccgac)
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“37 INP-SEQ 3” (ggtgtggaagccgacattc)
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The plasmid insert was found to be exactly as desired.  Highlighted below is the MCS excerpt from the “G101” primer results (“G101” is a reverse primer which is why the sequence is in reverse complement).
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Conclusions
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The construct sequence was produced exactly as designed; the full FLAG-tag – with BglII and HindIII sites – is present at the N-terminus of the linker sequence after INP.  However, despite all this, possibly due to YFP’s C and N termini being on the same side of it, the FLAG-tag is not accessible to antibodies.

Revision as of 14:49, 4 October 2014

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1352006

User Reviews

UNIQ7763bd54d2ab3296-partinfo-00000000-QINU UNIQ7763bd54d2ab3296-partinfo-00000001-QINU


Restriction digest + Gel electrophoresis

Firgure 1; a Xba1 + HindIII restriction digest screen of recombinants. The recombinant which went on to become K1352006 is in lane 5. The “L” lane is DNA marker (ladder). The arrows are to highlight the distance travelled by the HindIII-negative recombinants.







Figure 2; a restriction digest verification of plasmid K1352006

For figure 2: the letters indicate the following:

L. 10,000bp – 500bp DNA marker “ladder”

N. K1352006 plasmid digested with no enzymes

E. K1352006 plasmid digested with EcoRI

X. K1352006 plasmid digested with XbaI

S. K1352006 plasmid digested with SpeI

P. K1352006 plasmid digested with PstI

EP. K1352006 plasmid digested with EcoRI and PstI

XS. K1352006 plasmid digested with XbaI and Spe1

DNA Sequencing The recombinant plasmid was Sanger-sequenced with the following sequencing primers. “G101” is a reverse primer, the rest are forward primers.

“G101” (attaccgcctttgagtgagc)

“G100” (tgccacctgacgtctaagaa)

“35 INP-SEQ 1” (ccgattcattaatgcagctgg)

“36 INP-SEQ 2” (gaggttgctgttgccgac)

“37 INP-SEQ 3” (ggtgtggaagccgacattc)

The plasmid insert was found to be exactly as desired. Highlighted below is the MCS excerpt from the “G101” primer results (“G101” is a reverse primer which is why the sequence is in reverse complement).


Conclusions The construct sequence was produced exactly as designed; the full FLAG-tag – with BglII and HindIII sites – is present at the N-terminus of the linker sequence after INP. However, despite all this, possibly due to YFP’s C and N termini being on the same side of it, the FLAG-tag is not accessible to antibodies.