User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base: Difference between revisions
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== | ==11/18/09== | ||
* | * Unfinished line item | ||
* ✓ Finished line item | |||
---- | |||
'''Assemblies''' | |||
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size | |||
# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size | |||
> Digests (Fermentas FD)<br> | |||
> | --> Specific notes<br> | ||
{| class="wikitable" border="0" cellspacing=" | {| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn table --> | ||
|- | |- | ||
| | | <u>Reagent</u> || <u>Volume</u> | ||
| | |||
|- | |- | ||
| | | DNA (plasmid) || up to 25 μL | ||
| | |||
| | |||
|- | |- | ||
| | | 10x buffer || 3.0 | ||
| | |||
| | |||
|- | |- | ||
| | | enzyme 1 || 1.0 | ||
| | |||
| | |||
|- | |- | ||
| | | enzyme 2 || 1.0 | ||
| | |- | ||
| | | dH<sub>2</sub>O || --- | ||
|- | |||
| || 30 μL --> 37°C/ ~30 min. | |||
|} | |||
> Measure conc.'s | |||
{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table --> | |||
|- | |||
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u> | |||
|- | |||
| 1. KAH81 (X/P) || 0.519 || 1.67 || 25.9 | |||
|- | |||
| 2. huPcCD (E/S) || 1.590 || 1.76 || 79.5 | |||
|- | |||
| 3. mPcCD (E/S) || 1.604 || 1.76 || 79.5 | |||
|- | |||
| 4. flyPcCD (E/S) || 0.903 || 1.66 || 45.2 | |||
|- | |||
| 5. fshPcCD (E/S) || 1.482 || 1.74 || 74.1 | |||
|- | |||
| 6. hCBX8 (E/S) || 0.691 || 1.74 || 34.5 | |||
|- | |||
| 7. flyPc (E/S) || 0.118 || 1.44 || 5.9 | |||
|- | |||
| 2. KAH74 (E/X) || 0.188 || 1.37 || 9.4 | |||
|} | |||
> Dephosphorylation (Roche) | |||
{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table --> | |||
|- | |||
| <u>Reagent</u> || <u>Volume</u> | |||
|- | |||
| DNA (clean digest) || up to 17 μL (500 ng) | |||
|- | |||
| 10x buffer d.p. || 2.0 | |||
|- | |||
| phosphatase || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL | |||
|} | |} | ||
< | > Ligations (*Note: use insert + old backbone in calculations) | ||
{| class="wikitable" border="0" cellspacing="3" | |||
| <u>Ligation</u> || <u>Plate results (lig : neg crtl)</u> 11/19/09 | |||
|- | |||
| 1. KAH81(X/P)/2640, 33 ng + KAH48(S/P)/~3200, 20 ng || KAH86 >10:1 (Pick 2) | |||
|- | |||
| 2. KAH48(S/P)/ 20 ng || | |||
|- | |||
| 3. huPcCD(E/S)/3386*, > 42 ng + KAH74(E/X)/4061, 25 ng || KAH87 >10:1 (Pick 2) | |||
|- | |||
| 4. mPcCD(E/S)/3386*, > 42 ng + " || KAH88 >10:1 (Pick 2) | |||
|- | |||
| 5. flyPcCD(E/S)/3386*, > 42 ng + " || KAH89 >10:1 (Pick 2) | |||
|- | |||
| 6. fshPcCD(E/S)/3386*, > 42 ng + " || KAH90 >10:1 (Pick 2) | |||
|- | |||
| 7. hCBX8(E/S)/1170, > 42 ng + " || KAH91 >10:1 (Pick 2) | |||
|- | |||
| 8. hCBX8(E/S)/1170, > 42 ng + " || KAH92 >10:1 (Pick 2) | |||
|- | |||
| 9. KAH74(S/P)/ 20 ng || | |||
|} | |} | ||
{| class="wikitable" border="0" cellspacing="3" | |||
| || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9 | |||
|- | |||
| Insert DNA || 1.4 || --- || 1.7 || 1.7 || 1.7 || 1.7 || 0.5 || 2.5 || --- | |||
|- | |||
| Vector DNA || 0.6 || 0.6 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || --- | |||
|- | |||
| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 | |||
|- | |||
| T4 ligase (NEB) || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 | |||
|- | |||
| dH<sub>2</sub>O || 2.0 || 3.4 || 0.3 || 0.3 || 0.3 || 0.3 || 1.5 || --- || 2.0 | |||
|- | |||
| || 10 μL || 10 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11.5 μL || 11 μL | |||
|} | |||
---- | |||
'''Oligo annealing''' | |||
# NLS myc stop (KAH011) | |||
# K-stop (KAH012) | |||
# Adapter BglII/EcoRI | |||
# Adapter PstI/BglII | |||
{| class="wikitable" border="0" cellspacing="3" | |||
| DNA (oligos, 100 μM) || up to 18 μL | |||
|- | |||
| 10x annealing buffer || 2.0 | |||
|- | |||
| dH<sub>2</sub>O || --- | |||
|- | |||
| || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight | |||
|} | |||
Revision as of 07:13, 20 November 2009
Project name | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
11/18/09
Assemblies
> Digests (Fermentas FD)
> Ligations (*Note: use insert + old backbone in calculations)
Oligo annealing
|