Difference between revisions of "Part:BBa K118008:Experience"
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<br>GC content: 46.67%&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Location: 168-198
 
<br>GC content: 46.67%&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Location: 168-198
 
<br>Melting temp: 79.0°C&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Mismatched bases: 1
 
<br>Melting temp: 79.0°C&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Mismatched bases: 1
−
<br>Length: 30 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Mutation: Deletion
+
<br>Length: 30 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Mutation: Deletion
−
<br>5' flanking region: 15 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Forward primer MW: 9206.10 Da
+
<br>5' flanking region: 15 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; ;Forward primer MW: 9206.10 Da
−
<br>3' flanking region: 15 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Reverse primer MW: 9206.10 Da
+
<br>3' flanking region: 15 bp&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; Reverse primer MW: 9206.10 Da
  
 
<br>2.Find the best PCR condition by gradient PCR
 
<br>2.Find the best PCR condition by gradient PCR
 
<br>3.KOD PCR condition
 
<br>3.KOD PCR condition
 
<br>https://static.igem.org/mediawiki/parts/6/6c/PCR_condition.jpg
 
<br>https://static.igem.org/mediawiki/parts/6/6c/PCR_condition.jpg
 +
<br>4.Confirm the PCR product with electrophoresis
 +
5.DPN1 37℃ for 3hr~overnight
 +
<br>https://static.igem.org/mediawiki/parts/6/68/DPN1.jpg
 +
<br>6.80℃ 20mins to denature the DPN1
 +
<br>7.Confirm with electrophoresis
 +
<br>8.Self ligation room temperature for 2~3 hr
 +
<br>https://static.igem.org/mediawiki/parts/1/15/Self_ligation.jpg
 +
<br>9.Transform DH5alpha with the self-ligation product
 +
<br>Self-ligation product 20 ul
 +
<br>DH5alfa 50 ul
 +
<br>10.Incubate in Ap25 plate then transfer to Ap50 plate.
 +
 +
<br> We correct the Spe1 perfectly.
 +
Following is the gel electrophoresis result that show the uncut plasmid and the plasmid cut by Spe1.
 +
<br>https://static.igem.org/mediawiki/parts/a/a5/Crty_mutate-2.png

Revision as of 05:36, 7 October 2011

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_K118008

User Reviews

UNIQ74abd44309617c2e-partinfo-00000000-QINU UNIQ74abd44309617c2e-partinfo-00000001-QINU

Characterisation by 2011 iGEM NCTU_FORMOSA

We found a point mutation of Part:BBa_K118008 (iGEM08_Edinburgh) at Spe1 site and we corrected it successfully.(ACCTAGT->ACTAGT) This corrected part is numbered Part:BBa_K539119 The following is the gel electrophoresis result of crtY Part:BBa_K118008. The sample included are uncut plasmid, cut by EcoR1, Xba1, Spe1 and Pst1 respctivly.
Crty_mutate-1.png
We can see that the location of plasmid cut by Spe1 is the same as uncut plasmid. We assumed that Spe1 sit has mutated, so we made the sequence of gene of crtY. Following is the original sequence of crtY Part:BBa_K118008. The SpeI mutate into ACCTAGT.
Crty_mutate--3.jpg
Therefore, it need to delete the additional amino acid,C. The protocol we used is shown below.
Point Mutation
1.Design primers
Crty_mutate-4.jpg
GC content: 46.67%                              Location: 168-198
Melting temp: 79.0°C                              Mismatched bases: 1
Length: 30 bp                                      Mutation: Deletion
5' flanking region: 15 bp                           ;Forward primer MW: 9206.10 Da
3' flanking region: 15 bp                           Reverse primer MW: 9206.10 Da


2.Find the best PCR condition by gradient PCR
3.KOD PCR condition
PCR_condition.jpg
4.Confirm the PCR product with electrophoresis 5.DPN1 37℃ for 3hr~overnight
DPN1.jpg
6.80℃ 20mins to denature the DPN1
7.Confirm with electrophoresis
8.Self ligation room temperature for 2~3 hr
Self_ligation.jpg
9.Transform DH5alpha with the self-ligation product
Self-ligation product 20 ul
DH5alfa 50 ul
10.Incubate in Ap25 plate then transfer to Ap50 plate.


We correct the Spe1 perfectly. Following is the gel electrophoresis result that show the uncut plasmid and the plasmid cut by Spe1.
Crty_mutate-2.png