Haynes Lab:Notebook/Jan/2015/03/27: Difference between revisions

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==03/27/15==
==03/27/15==
* Cultures - BD006/MV2 colonies (from 3/26/15 ligations), 5 mL each (4 total)
* Cultures - BD006/MV2 colonies (from 3/26/15 ligations), 5 mL each (4 total)
* Plasmid minipreps - DPRE cultures (5 mL each)
* Plasmids - DPRE minipreps (5 mL cultures)
 
----
'''Plasmids - DPRE minipreps'''
*'''[[User:Karmella Haynes|---Karmella]] 15:12, 27 March 2015 (EDT)''':
* Sigma GenElute Miniprep kit.
* Elute with 70μL elution buffer
# DPRE a
# DPRE b
 
* Digests (Fermentas FD)
** E/S to prep for insertion upstream of BD004
** Also digest BD004 w/ E/X
 
{| {{table}} cellspacing="3" <!-- Digest rxn. table -->
|- valign="top"
| bgcolor=#cfcfcf | Reagent
| bgcolor=#cfcfcf | Volume
| rowspan="7" | Expected:<br>1,2. DPRE (E/S) = ###, 3200<br>3. BD004 (E/X) = 7318
| rowspan="7" | [[Image:KAH032015_gel1.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder]
|-
| DNA (plasmid) || 20.0 μL
|-
| 10x buffer || 3.0
|-
| enzyme 1 || 1.0
|-
| enzyme 2 || 1.0
|-
| dH<sub>2</sub>O || ---
|-
| &nbsp; || 30 μL --> 37°C/ ~30 min.
|}
 
 
* Conclusions
** MV2 is correct
** BD004 - most likely missing the D11.12 fragment. Sequence results from Meli'sa's past work agrees with the short fragment size on the gel.
** BD006  is correct
 
* Miniprep conc.'s
** Combined the pairs of preps. Pairs look identical.
{| {{table}} cellspacing="3" <!-- [DNA] table -->
|- bgcolor=#cfcfcf
| Sample || OD260 || 260/280 || ng/μL
|-
| 1. MV2 mp || 0.132 || 1.85 || 132.262
|-
| 2. BD004 mp || 0.144 || 1.91 || 143.967
|-
| 3. BD006 mp || 0.078 || 1.83 || 78.413
|}




Revision as of 12:12, 27 March 2015

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03/27/15

  • Cultures - BD006/MV2 colonies (from 3/26/15 ligations), 5 mL each (4 total)
  • Plasmids - DPRE minipreps (5 mL cultures)

Plasmids - DPRE minipreps

  • ---Karmella 15:12, 27 March 2015 (EDT):
  • Sigma GenElute Miniprep kit.
  • Elute with 70μL elution buffer
  1. DPRE a
  2. DPRE b
  • Digests (Fermentas FD)
    • E/S to prep for insertion upstream of BD004
    • Also digest BD004 w/ E/X
Reagent Volume Expected:
1,2. DPRE (E/S) = ###, 3200
3. BD004 (E/X) = 7318 		Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O ---
  30 μL --> 37°C/ ~30 min.


  • Conclusions
    • MV2 is correct
    • BD004 - most likely missing the D11.12 fragment. Sequence results from Meli'sa's past work agrees with the short fragment size on the gel.
    • BD006 is correct
  • Miniprep conc.'s
    • Combined the pairs of preps. Pairs look identical.
Sample OD260 260/280 ng/μL
1. MV2 mp 0.132 1.85 132.262
2. BD004 mp 0.144 1.91 143.967
3. BD006 mp 0.078 1.83 78.413



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