User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/23: Difference between revisions
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<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* Ryan - Receiver cloning stage 2 | * Ryan - Receiver cloning stage 2 | ||
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* Digests (Fermentas FD) | * Digests (Fermentas FD) | ||
** E/X | ** E/X | ||
** Inserts are gBlocks = 2.0 ng/μL | |||
{| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table --> | {| {{table}} cellspacing="3" <!-- Digest for dephos rxn. table --> | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA ( | | DNA (20 ng) || 10.0 μL | ||
|- | |- | ||
| 10x buffer || 2.0 | | 10x buffer || 2.0 | ||
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| XbaI || 1.0 | | XbaI || 1.0 | ||
|- | |- | ||
| dH<sub>2</sub>O || | | dH<sub>2</sub>O || 6.0 | ||
|- | |- | ||
| || 20.0 μL --> 37°C/ ~15 min. | | || 20.0 μL --> 37°C/ ~15 min. | ||
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| bgcolor=#cfcfcf | Volume | | bgcolor=#cfcfcf | Volume | ||
|- | |- | ||
| DNA (digest) || 20.0 μL ( | | DNA (digest) || 20.0 μL (20 ng) | ||
|- | |- | ||
| phosphatase || 1.0 | | phosphatase || 1.0 | ||
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| dH<sub>2</sub>O || --- | | dH<sub>2</sub>O || --- | ||
|- | |- | ||
| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~ | | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = ~1 ng/μL | ||
|} | |} | ||
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* Ligation/Digestion Reactions | * Ligation/Digestion Reactions | ||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert: | ** Inserts are gBlocks = ~2ng/μL | ||
** For an insert = ~200 and vector = ~4000, need 5 ng insert for 2:1 insert:vector(50 ng) ratio | |||
{| {{table}} cellspacing="3" <!-- Lig/Dig rxn table --> | {| {{table}} cellspacing="3" <!-- Lig/Dig rxn table --> | ||
| || 1 || 2 || | | || 1 || 2 || | ||
|- | |- | ||
| Insert DNA || | | Insert DNA || 5.0 || --- || | ||
|- | |- | ||
| Vector DNA || ### || ### || | | Vector DNA || ### || ### || | ||
|- | |- | ||
| 2x lgn buf (Roche) || | | 2x lgn buf (Roche) || 7.5 || ### || | ||
|- | |- | ||
| T4 ligase (NEB) || 1.0 || 1.0 || | | T4 ligase (NEB) || 1.0 || 1.0 || | ||
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| dH<sub>2</sub>O || ### || ### || | | dH<sub>2</sub>O || ### || ### || | ||
|- | |- | ||
| || | | || 15.0 μL || # μL || | ||
|} | |} | ||
Thermal cycler: Bio-Rad C1000 | |||
* | |||
Revision as of 16:38, 23 April 2015
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
04/23/15
Ryan - Receiver plasmids, assembly
Thermal cycler: Bio-Rad C1000
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