Part:BBa_K496000

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]