User:Kristina M Holton/Notebook

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2.7.2007 Monday</dt>
miRNA Extraction using 1st protocol and Qiagen miRNEasy Kit

Large Fibroma -> very very fatty

Used 3ml TRIzol from Invitrogen

While standing, TRIzol and fat start to pellet at the bottom; removed supernatants and centrifuged both pellet and supernatant

After centrifugation, already appeared to be phase separation

Still added glycogen + chloroform, stand RT for 10min then centrifuge 10000g 20min

Added EtOH: large amount of white, "SDS-like" precipitate

Very difficult to add precipitate to RNeasy Mini spin columns, even with additional EtOH


Differences in 2nd protocol (probable fixes):

Use 2ml QIAzol for lysis

If: no debris accumulates and the QIAzol remains pink

Skip first centrifugation step + removing the supernatant

Else: (Brown QIAzol or debris accumulation)

Centrifuge and remove supernatant

Try to add all precipitate from EtOH step</dd>

3.7.2007 Tuesday</dt>
DNA MaxiPrep C6-C10

Someone turned off shaker in warm room -> cell culture not as turbid as it should be

Checked overnight culture's OD600 at 9:45 -> .369 (need at least .5, .6-.7 desirable)

P1 Buffer: need to bring stock Tris-HCl pH 7.0 up to 8.0; need RNase A

Cells spun down ~ 4:30, stored in 4C overnight


miRNA Extraction and Purification Using 2nd Protocol

Patient: DR

2ml might not be enough QIAzol for tissue grinder -> increased to about 3ml

Did not need first centrifuge step

Good yield RNA (~24ug)</dd>

4.7.2007 Wednesday</dt>
No entries for this date. Please feel free to add entries.</dd>
5.7.2007 Thursday</dt>
No entries for this date. Please feel free to add entries.</dd>
6.7.2007 Friday</dt>
No entries for this date. Please feel free to add entries.</dd>
7.7.2007 Saturday</dt>
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8.7.2007 Sunday</dt>
No entries for this date. Please feel free to add entries.</dd>

9.7.2007 Monday</dt>
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10.7.2007 Tuesday</dt>
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11.7.2007 Wednesday</dt>
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12.7.2007 Thursday</dt>
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13.7.2007 Friday</dt>
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14.7.2007 Saturday</dt>
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15.7.2007 Sunday</dt>
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