Haynes Lab:Notebook/HPK-CFP insertion into Gal4EED/Luc using CRISPR/2015/04/16: Difference between revisions

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Also, for future reference: when performing digestion/ligation, set up a control plate using linearized vector treated with phosphatase. Presence of colonies on this plate indicates that the phosphatase step did not completely work.
Also, for future reference: when performing digestion/ligation, set up a control plate using linearized vector treated with phosphatase. Presence of colonies on this plate indicates that the phosphatase step did not completely work.


-----
<b>--DNA Concentration--</b>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample ID'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/µL)'''
| align="center" style="background:#f0f0f0;"|'''St Dev (ng/µL)'''
|-
| DBN001_pSB1A3, colony 1||56.7||2.6
|-
| DBN001_pSB1A3, colony 2||54.0||0.2
|}
-----
<b>--Restriction Digest--</b>
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Reagent'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| DNA||600ng (12 µL)
|-
| 10x FastDigest Buffer||3
|-
| EcoRI||1
|-
| PstI||1
|-
| Water||13
|-
| Total||30
|}
Digest for 15 minutes at 37°C.
<b>NOTE:</b> Should've used SpeI, not PstI. Oh well, won't make much of a difference for analysis of the sequence.
-----
<b>--Gel Electrophoresis--</b>
Expected size fragments from EcoRI/PstI digestion: 2114, 189, 53


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Revision as of 13:37, 16 April 2015

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Plasmid Miniprep of gBlock in mCherry vector

--Notes--

Both colonies grown in liquid culture are normal colored (not red). Cell density appears low though, with lots of sticky filaments. Indication that they were not subjected to continuous shaking overnight.

Also, for future reference: when performing digestion/ligation, set up a control plate using linearized vector treated with phosphatase. Presence of colonies on this plate indicates that the phosphatase step did not completely work.


--DNA Concentration--

Sample ID Conc. (ng/µL) St Dev (ng/µL)
DBN001_pSB1A3, colony 1 56.7 2.6
DBN001_pSB1A3, colony 2 54.0 0.2

--Restriction Digest--

Reagent Volume
DNA 600ng (12 µL)
10x FastDigest Buffer 3
EcoRI 1
PstI 1
Water 13
Total 30

Digest for 15 minutes at 37°C.

NOTE: Should've used SpeI, not PstI. Oh well, won't make much of a difference for analysis of the sequence.


--Gel Electrophoresis--


Expected size fragments from EcoRI/PstI digestion: 2114, 189, 53


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