Difference between revisions of "Part:BBa K349119"
Rpgguardian (Talk | contribs) |
(→Usage and Biology) |
||
| (4 intermediate revisions by one other user not shown) | |||
| Line 6: | Line 6: | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
| − | This part is compatible with the Team Alberta 2010 Biobytes 2.0 assembly method. It can be utilized as an AB BioByte following digestion with | + | This part is compatible with the Team Alberta 2010 Biobytes 2.0 assembly method. It can be utilized as an AB BioByte following digestion with Bfu AI and purification. The following protocol outlines the Biobytes 2.0 assembly method: <br> |
| − | 1) Mix the iron micro beads for 10 minutes. | + | 1) Mix the iron micro beads for 10 minutes.<br> |
| − | 2) Transfer 20 ul of iron micro beads to a 1.5 mL tube. | + | 2) Transfer 20 ul of iron micro beads to a 1.5 mL tube. <br> |
| − | 3) Pull the beads to the side using a magnetic tube rack. | + | 3) Pull the beads to the side using a magnetic tube rack. <br> |
| − | 4) Remove and discard the supernatant. | + | 4) Remove and discard the supernatant. <br> |
| − | 5) Add 50 ul of BioBytes 2.0 Wash Buffer to the beads. Flick gently to resuspend. | + | 5) Add 50 ul of BioBytes 2.0 Wash Buffer to the beads. Flick gently to resuspend. <br> |
| − | 6) Pull the beads to the side using a magnetic tube rack. | + | 6) Pull the beads to the side using a magnetic tube rack. <br> |
| − | 7) Remove and discard the supernatant. | + | 7) Remove and discard the supernatant. <br> |
| − | 8) Repeat steps 5 to 7. | + | 8) Repeat steps 5 to 7. <br> |
| − | 9) Add 200 ng of a premade anchor-Byte construct (premade in the GENOMIKON kit) to the beads and top off the volume to 20ul with TE Buffer. Flick gently to resuspend. | + | 9) Add 200 ng of a premade anchor-Byte construct (premade in the GENOMIKON kit) to the beads and top off the volume to 20ul with TE Buffer. Flick gently to resuspend. <br> |
| − | 10) Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube. | + | 10) Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube. <br> |
| − | 11) Repeat steps 6 and 7. | + | 11) Repeat steps 6 and 7. <br> |
| − | 12) Repeat steps 5 to 7. | + | 12) Repeat steps 5 to 7. <br> |
| − | 13) Add 200 ng of the next BioByte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 20ul. | + | 13) Add 200 ng of the next BioByte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 20ul. <br> |
| − | 14) Flick gently to resuspend. Allow ligation for five minutes, flicking gently every minute. | + | 14) Flick gently to resuspend. Allow ligation for five minutes, flicking gently every minute. <br> |
| − | 15) Add 30ul of Wash Buffer to the tube. Flick gently. | + | 15) Add 30ul of Wash Buffer to the tube. Flick gently. <br> |
| − | 16) Repeats steps 6 and 7. | + | 16) Repeats steps 6 and 7. <br> |
| − | 17) Repeat steps 5 to 7 twice. | + | 17) Repeat steps 5 to 7 twice. <br> |
| − | 18) Repeat steps 13 to 17 for each subsequent Byte addition, including the last BioByte. | + | 18) Repeat steps 13 to 17 for each subsequent Byte addition, including the last BioByte. <br> |
| − | 19) After the last Byte has been ligated and washed, add 30 ul of 75C Elution Buffer. Keep at 75C for 10 minutes. | + | 19) After the last Byte has been ligated and washed, add 30 ul of 75C Elution Buffer. Keep at 75C for 10 minutes. <br> |
| − | 20) After 10 minutes, still at 75C, put the tube into a magnetic rack . Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct. | + | 20) After 10 minutes, still at 75C, put the tube into a magnetic rack . Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct. <br> |
| + | |||
| + | For more information on this construction method click <html><a href="http://2010.igem.org/Team:Alberta/biobyte2">here</a></html> | ||
<!-- --> | <!-- --> | ||
Latest revision as of 17:35, 11 August 2011
mWasabi-AB (BioBytes 2.0)
Wasabi Fluorescent Protein. Please note this part is "naked" meaning it does not contain a promoter or RBS, it is also lacking start and stop codons. The complete ORF is intended to be reconstituted using BA linker Bytes in the BioBytes 2.0 assembly method.
Usage and Biology
This part is compatible with the Team Alberta 2010 Biobytes 2.0 assembly method. It can be utilized as an AB BioByte following digestion with Bfu AI and purification. The following protocol outlines the Biobytes 2.0 assembly method:
1) Mix the iron micro beads for 10 minutes.
2) Transfer 20 ul of iron micro beads to a 1.5 mL tube.
3) Pull the beads to the side using a magnetic tube rack.
4) Remove and discard the supernatant.
5) Add 50 ul of BioBytes 2.0 Wash Buffer to the beads. Flick gently to resuspend.
6) Pull the beads to the side using a magnetic tube rack.
7) Remove and discard the supernatant.
8) Repeat steps 5 to 7.
9) Add 200 ng of a premade anchor-Byte construct (premade in the GENOMIKON kit) to the beads and top off the volume to 20ul with TE Buffer. Flick gently to resuspend.
10) Allow annealing for 30 minutes, mixing by flicking every 5 minutes. Ensure that there are no droplets on the sides of the tube.
11) Repeat steps 6 and 7.
12) Repeat steps 5 to 7.
13) Add 200 ng of the next BioByte of your construct, making sure that a BA Byte follows an AB Byte and vice versa. Add an appropriate amount of 2x QuickLigase Buffer, Quick Ligase and TE to a total volume of 20ul.
14) Flick gently to resuspend. Allow ligation for five minutes, flicking gently every minute.
15) Add 30ul of Wash Buffer to the tube. Flick gently.
16) Repeats steps 6 and 7.
17) Repeat steps 5 to 7 twice.
18) Repeat steps 13 to 17 for each subsequent Byte addition, including the last BioByte.
19) After the last Byte has been ligated and washed, add 30 ul of 75C Elution Buffer. Keep at 75C for 10 minutes.
20) After 10 minutes, still at 75C, put the tube into a magnetic rack . Allow the beads to be pulled aside and collect the supernatant into a clean 1.5 mL tube. The supernatant will contain your construct.
For more information on this construction method click here
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 27
Illegal BsaI.rc site found at 438