User:Karmella Haynes/Notebook/BioBrick cloning/Entry Base

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(mm/dd/yy)
(11/18/09)
Line 41: Line 41:
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
|-
|-
-
| 1. KAH81 (X/P) || 0.519 || 1.67 || 25.9
+
| 1. Digested part (a/b) || --- || --- || ---
|-
|-
-
| 2. huPcCD (E/S) || 1.590 || 1.76 || 79.5
+
| 2. Digested part (c/d) || --- || --- || ---
-
|-
+
-
| 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
+
|}
|}
Line 76: Line 64:
-
 
+
> Ligations
-
> Ligations (*Note: use insert + old backbone in calculations)
+
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
-
{| class="wikitable" border="0" cellspacing="3"
+
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
-
| <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)
+
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick x:y (Pick 2#)</font>
|-
|-
-
| 2. KAH48(S/P)/ 20 ng || &nbsp;
+
| 2. vector(c/d)/ ## ng || &nbsp;
-
|-
+
-
| 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 || &nbsp;
+
|}
|}
-
{| class="wikitable" border="0" cellspacing="3"
+
{| class="wikitable" border="0" cellspacing="3" <!-- Ligantion rxn table -->
-
| &nbsp; || 1 || 2 || 3 || 4 || 5 || 6 || 7 || 8 || 9
+
| &nbsp; || 1 || 2 ||
|-
|-
-
| Insert DNA        || 1.4 || --- || 1.7 || 1.7 || 1.7 || 1.7 || 0.5 || 2.5 || ---
+
| Insert DNA        || ### || --- ||
|-
|-
-
| Vector DNA        || 0.6 || 0.6 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || 2.5 || ---
+
| Vector DNA        || ### || ### ||
|-
|-
-
| 2x lgn buf (Roche) || 5.0 || 5.0 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5 || 5.5
+
| 2x lgn buf (Roche) || ### || ### ||
|-
|-
-
| T4 ligase (NEB)    || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0 || 1.0
+
| T4 ligase (NEB)    || 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
+
| dH<sub>2</sub>O    || ### || ### ||
|-
|-
-
| &nbsp; || 10 μL || 10 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11 μL || 11.5 μL || 11 μL
+
| &nbsp;             || # μL || # μL ||
|}
|}
----
----
'''Oligo annealing'''
'''Oligo annealing'''
-
# NLS myc stop (KAH011)
+
# New BB 1
-
# K-stop (KAH012)
+
# New BB 2
-
# Adapter BglII/EcoRI
+
-
# Adapter PstI/BglII
+
-
 
+
-
{| class="wikitable" border="0" cellspacing="3"
+
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
-
| DNA (oligos, 100 μM) || up to 18 μL
+
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
|-
| 10x annealing buffer || 2.0
| 10x annealing buffer || 2.0

Revision as of 09:33, 20 November 2009

Project name Main project page

11/18/09

  • Unfinished line item
  • ✓ Finished line item

Assemblies

  1. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
  2. BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size

> Digests (Fermentas FD)
--> Specific notes

Reagent Volume
DNA (plasmid) up to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


> Measure conc.'s

Sample OD260 260/280 ng/μL
1. Digested part (a/b) --- --- ---
2. Digested part (c/d) --- --- ---


> Dephosphorylation (Roche)

Reagent Volume
DNA (clean digest) up to 17 μL (500 ng)
10x buffer d.p. 2.0
phosphatase 1.0
dH2O ---
  20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng new BioBrick x:y (Pick 2#)
2. vector(c/d)/ ## ng  
  1 2
Insert DNA ### ---
Vector DNA ### ###
2x lgn buf (Roche) ### ###
T4 ligase (NEB) 1.0 1.0
dH2O ### ###
  # μL # μL

Oligo annealing

  1. New BB 1
  2. New BB 2
DNA (oligos, 100 μM) up to 18 μL (3 μL ea.)
10x annealing buffer 2.0
dH2O ---
  20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight



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