Difference between revisions of "Part:BBa K496000"

Revision as of 15:02, 1 October 2011

Forward primer for visualizing restriction enzyme digestion.

Fig. 1

Fig. 2

Sometimes standard protocol fails to produce plasmid, or BioBrick is toxic to host cell and can't be amplified. In moments like these, PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.

When we amplify BioBrick parts or vectors, primers which anneal to prefix and suffix are used, because they are universal. But unlike miniprep products, PCR products don't produce two distinct bands when they are digested. So we are left wondering if digestion went well.

The iGEM HokkaidoU 2010 created a set of primers which solves this problem. The idea is quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the Biobrick (Fig. 1).

PCR products that are amplified by using this primer set produces distinct approx 100 bp and 200 bp fragments (Fig. 2).

You can judge if digestion was successful by presence of these bands. Compared to the intact PCR product, you will see the significant change in digested PCR fragment length. The fact that all 10 current high copy number assembly vectors containing pSB1C3 share the same annealing sites in the BioBrick flanking region makes this possible.

This primer set makes amplified BioBricks 300bp longer. So, this makes small parts like RBS easier to handle. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.

Works with

*More...*
Name	Description	Resistance	Replicon	Copy number	Chassis	Length
BBa_J428353	pJUMP28-1A KanR Type IIS Level 1 vector. Origin pUC (high copy number)	kanamycin				2524
BBa_K1362091	pSB1A30: High copy BioBrick cloning/expression backbone carrying Amp resitance	A	pMB1	100-300		2206
BBa_K5194000	pSB1K0 Yeast Shuttle Vector (sfGFP dropout) Type IIS	Kanamycin	Origin pUC (high copy number)		Pichia pastoris	5355
BBa_K823055	pSB1C3F-Vector pSB1C3 with RFP-cassette in Freiburg Standard	C	pMB1	100-300		2102
pSB1A3	High copy BioBrick assembly plasmid	A	pMB1	100-300		2155
pSB1A7	Transcriptionally insulated high copy BioBrick plasmid	A	pMB1	100-300		2431
pSB1AC3	High copy BioBrick assembly plasmid	AC	pMB1	100-300		3055
pSB1AK3	High copy BioBrick assembly plasmid	AK	pMB1	100-300		3189
pSB1AT3	High copy BioBrick assembly plasmid	AT	pMB1	100-300		3446
pSB1C3	High copy BioBrick assembly plasmid	C	pMB1	100-300		2070
pSB1K3	High copy BioBrick assembly plasmid	K	pMB1	100-300		2204
pSB1T3	High copy BioBrick assembly plasmid	T	pMB1	100-300		2461

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K496000 short</partinfo>
-Sometimes standard protocol fails to produce plasmid or BioBrick is toxic to host cell and can't by amplified. In moments like these PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.
+[[Image:HokkaidoU_Japan_PCR_Protocol_Fig1.png|right|400px|thumb|Fig. 1]]
+[[Image:HokkaidoU_Japan_PCR_Protocol_Fig2.png|right|150px|thumb|Fig. 2]]
+&nbsp;&nbsp;&nbsp;Sometimes standard protocol fails to produce plasmid, or BioBrick is toxic to host cell and can't be amplified. In moments like these, PCR is handy. PCR is also great because it doesn't have many steps, the more steps the more ways to fail.
-<br>
+&nbsp;&nbsp;&nbsp;When we amplify BioBrick parts or vectors, primers which anneal to prefix and suffix are used, because they are universal. But unlike miniprep products, PCR products don't produce two distinct bands when they are digested. So we are left wondering if digestion went well.
-When amplifying BioBricks primers which anneal to prefix and suffix are used. Good thing about them is that they are universal so with only 2 you can amplify all BioBricks. But unlike miniprep product BioBrick amplified like this don't produce 2 distinct bands when digested. So you are left wondering if digestion went well.
+&nbsp;&nbsp;&nbsp;The iGEM HokkaidoU 2010 created a set of primers which solves this problem. The idea is quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the Biobrick (Fig. 1).
-<br>
+&nbsp;&nbsp;&nbsp;PCR products that are amplified by using this primer set produces distinct approx 100 bp and 200 bp fragments (Fig. 2).
-We created a set of primers which solves this problem. You can judge if digestion was successful by presence of approximatively 100 bp or/and 200 bp bands and significant change in PCRed fragments length.
+&nbsp;&nbsp;&nbsp;You can judge if digestion was successful by presence of these bands. Compared to the intact PCR product, you will see the significant change in digested PCR fragment length. The fact that all 10 current high copy number assembly vectors containing pSB1C3 share the same annealing sites in the BioBrick flanking region makes this possible.
-<br>
+&nbsp;&nbsp;&nbsp;This primer set makes amplified BioBricks 300bp longer. So, this makes small parts like RBS easier to handle. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.
-So idea behind this primers quite simple. We just designed primers which anneal 100 bp upstream and 200 bp down stream from the part like in Fig.1 bellow.
-<br>
-[[Image:HokkaidoU_Japan_PCR_Protocol_Fig1.png|Fig.1]]
-<br>
-[[Image:HokkaidoU_Japan_PCR_Protocol_Fig2.png|thumb|Fig.2]]
-Now when digested PCRed fragment produces distinct approx 100 bp or/and 200 bp fragment depending on cut sites. Knowing this you can continue to the next step with the sure and light heart (see Fig.2). This is crucial when you take into account that many participants have limited at the bench experience before hand.
-<br>
-As everyone have guest by now these primers anneal to plasmid regions flanking the BioBrick. And there are more than 40 different primers. But not all of them are quite so different in fact all 7 registries assembly plasmids share the same sequence on the BioBrick flanking regions. And few others do also, bringing the number up to about 10.
-<br>
-And with the new registry requirement to submit all BioBricks in pSB1C3 plasmid present the possibility that in near future many if not all plasmids could be supported by this primers making assembly easier.
-This primer set makes amplified BioBricks 300bp longer making small parts like RBS easier to extract from gel. And even when small BioBrick is single enzyme digested it is still longer than 100bp or 200bp accordingly. This primer set is currently experimental.
+<div style="clear:both"></div>
 Works with
-With assembly plasmids
+<parttable>high_copy_biobrick_assembly_plasmid</parttable>
-pSB1A3
-pSB1A7
-pSB1AC3
-pSB1AK3
-pSB1AT3
-pSB1C3
-pSB1K3
-pSB1T3
-Biobricks PCRed with these primers produce fragment approximatively  100 bp and/or 200bp when digested with restriction enzymes.
-Fragment lenght
-Does not work with, because one of the primers site is missing:
-<br>
-pSB2K3
 <!-- Add more about the biology of this part here