User:Karmella Haynes/Notebook/BioBrick cloning/2015/04/16: Difference between revisions

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| Sample || OD260 || 260/280 || ng/μL
| Sample || OD260 || 260/280 || ng/μL
|-
|-
| 1. MV10 XbaI || --- || --- || ---
| 1. MV10 XbaI || 0.017 || 1.47 || 17.24
|}
|}




* Dephosphorylation (Roche)
{| {{table}} cellspacing="3" <!-- Dephos table -->
|-
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
|-
| DNA (clean digest) || up to 17 μL (500 ng)
|-
| 10x buffer d.p. || 2.0
|-
| phosphatase || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|}
* Ligations
{| {{table}} cellspacing="3" <!-- Ligations table -->
|- bgcolor=#cfcfcf
| Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
| 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
|-
| 2. vector(c/d)/ ## ng || &nbsp;
|}
{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
| &nbsp;            || 1    || 2    ||
|-
| Insert DNA        || ###  || ---  ||
|-
| Vector DNA        || ###  || ###  ||
|-
| 2x lgn buf (Roche) || ###  || ###  ||
|-
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
| dH<sub>2</sub>O    || ###  || ###  ||
|-
| &nbsp;            || # μL || # μL ||
|}
----
'''Oligo annealing'''
# New BB 1
# New BB 2
{| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
| DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
|-
| 10x annealing buffer || 2.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
|}




Revision as of 11:51, 16 April 2015

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04/16/15

  • LCR Development - PCR MV10 bacbone


LCR Development

  • Try PCR on linearized vector
Reagent Volume Expected:
1. BB 1 = size
2. BB2 = size
DNA(plasmid) 5.0 μL
10X buffer 3.0
XbaI 2.0
dH2O 20.0
  30 μL --> 37°C/ ~30 min.


Measure conc.

  • DNA purified with Sigma PCR clean-up kit
  • Eluted& back-eluted with 30 μL elution sln.
Sample OD260 260/280 ng/μL
1. MV10 XbaI 0.017 1.47 17.24




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