Part:BBa_K530026:Design

GME

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 86
  Illegal BglII site found at 727
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Part was constructed by ordering individual oligonucleotides. These oligos were then assembled through the use of CPEC and then further amplified with the outermost oligos as primers. A PCR was then carried out. The PCR reaction performed also served to add the biobrick prefix and suffix to the terminator. PCR and tube contents This PCR was done with the use of Herculase Enzyme from Agilent.

Reagents	Volume (uL)
Herculase 5X Buffer	10
2.5mM dNTP	5
100-300ng DNA	X
10uM Forward Primer	1.25
10uM Reverse Primer	1.25
Herculase II Enzyme	1
Sterile Water	Till Total
Total	50

Temperature (C)	Time	Cycles
95	2 mins	1
95	20 secs	30
55	20 secs	30
72	30 secs	30
72	3 mins	1

5ul of the sample were run on a gel with loading dye. The resulting gel confirmed the product was of the right size (841bp) to be the insert with the biobrick prefix and suffix now attached.

The insert was then PCR purified and eluted with 10mM Tris HCl at ph7.4. Following the PCR purification, the inserts were digested with EcorI and SpeI.

Reagents	Volume (uL)
NEB Buffer 4	3
Insert	25
EcorI	1
SpeI	1
BSA	.3

The digestion mixture was then heat inactivated at 60 Celsius for 20 minutes. All 30uL were then loaded on a gel and gel extracted to ensure that the ssDNA removed by digestion doesn't religate. The gel piece was then gel purified through the use of Qiagen Gel Extraction Kit and eluted with 20uL Tris HCl at pH7.4.

Set the ligation overnight at 16 Celsius.

Reagents	Volume (uL)
10X T4 Lig. Buffer	1.6
Insert	6
Vector	3
T4 Ligase	1
Sterile Water	3.5

5uL of ligation product was transformed into DH5 Alpha Competent Cells through heat shock. Cells were re-suspended in 450uL LB and placed in shaker for an hour. Cells were then plated in chloroamphenicol. Chloroamphenicol plates were allowed to grow overnight at 37 Celsius. 10 Colonies were picked 12-16 hours later and grown up in LB with chloroamphenicol.

2uL Sample of colonies was placed in 50uL sterile water and allowed to heat to 95 Celsius to separate DNA from cell biomass. After 10 minutes, the lysed sample is placed on ice for 5 minutes. Sample is then ready for csPCR.

Reagents	Volume (uL)
Buffer	10
2.5mM dNTP	5
100-300ng DNA	X
10uM Forward Seq. Primer	1.25
10uM Reverse Seq. Primer	1.25
rTaq Polymerase	1.25
Sterile Water	Till Total
Total	25

Temperature (C)	Time	Cycles
95	2 mins	1
95	20 secs	25
55	20 secs	25
72	30 secs	25
72	3 mins	1

[[]]

Colonies 2, 4-5, 7-9 showed a band of the correct size.

Sent colony 2, 4, 7 and 8 for sequencing with the primers below:

Forward Primer: ccacctgacgtctaagaaac

Reverse Primer: tcactcaaaggcggtaatac

Sequencing results are as shown below.

[[]]

This is the sequencing for colony 7. Colony 7 had a perfect match with the genomic DNA promoter.

Source

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2034532/