Daniel:Notebook/ComboLock/2016-8-25: Difference between revisions

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<li>Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li>
<li>Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li>
<gallery perrow=2 heights=200px widths=200px>
<gallery perrow=2 heights=200px widths=200px>
File:2016-08-20-PosConTest-SizeSelect.png|Before Image
File:2016-08-25-PosConAmpTest2-SizeSelect.png|Before Image
File:2016-08-20-PosConTest-SizeSelect-After.png|After Image
File:2016-08-25-PosConAmpTest2-SizeSelect-After.png|After Image
</gallery>
</gallery>
<li>Centrifuge gel at 14000rpm for 1.5 minutes</li>
<li>Centrifuge gel at 14000rpm for 1.5 minutes</li>
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</ol>
</ol>


=Positive Control Amplicon Test=
=Positive Control Amplicon Test (Started [[Daniel:Notebook/ComboLock/2016-8-18|8/18/2016]])=




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|}
|}


Also below is the gel image from size selection. Note that index 11 didn't have any product of the right size, so I don't expect any results from that column.
<gallery perrow=2 heights=200px widths=200px>
File:2016-08-20-PosConTest-SizeSelect.png|Before Image
File:2016-08-20-PosConTest-SizeSelect-After.png|After Image
</gallery>
Now we can look at the results. In order to get these I used grep on the barcodes that are specific to each of the three positive control pieces (C1, C2 and C3). I grepped each barcode alone and in pairs with each other. Note that C1-C2 and C3-C2 are the expected products, C1-C3 should never exist.
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext>
|- style="background-color:#FCD5B4;font-size:12pt;font-weight:bold" align="center"
| width="180" height="37" | Name
| width="160" | Barcode Sequence
| width="65" | Index 10
| width="65" | Index 11
| width="65" | Index 12
| width="65" | Index 13
|- style="font-size:12pt"
| height="15"  valign="bottom" | Latch6 BC (C3)
|  valign="bottom" | GTTGGCAG
| align="center" align="center" valign="bottom" | 4
| align="center" align="center" valign="bottom" | 95
| align="center" align="center" valign="bottom" | 7658
| align="center" align="center" valign="bottom" | 11266
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Latch8 BC RC (C1)
|  valign="bottom" | TACAAATA
| align="center" align="center" valign="bottom" | 1140
| align="center" align="center" valign="bottom" | 187
| align="center" align="center" valign="bottom" | 14963
| align="center" align="center" valign="bottom" | 26222
|- style="font-size:12pt"
| height="15"  valign="bottom" | Latch9 BC RC (C2)
|  valign="bottom" | GTAGGCGC
| align="center" align="center" valign="bottom" | 1069
| align="center" align="center" valign="bottom" | 77
| align="center" align="center" valign="bottom" | 22059
| align="center" align="center" valign="bottom" | 37200
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Intersect L8-L9 (C1-C2)
|  valign="bottom" | TACAAATA,GTAGGCGC
| align="center" align="center" valign="bottom" | 1025
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 14047
| align="center" align="center" valign="bottom" | 25016
|- style="font-size:12pt"
| height="15"  valign="bottom" | Intersect (C3-C2)
|  valign="bottom" | GTTGGCAG,GTAGGCGC
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 2
| align="center" align="center" valign="bottom" | 7163
| align="center" align="center" valign="bottom" | 10759
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Intersect (C1-C3)
|  valign="bottom" | GTTGGCAG,TACAAATA
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 0
|- style="font-size:12pt"
| height="15"  valign="bottom" | Percent C2 Reads from Amplicons
| align="center"  valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | 95.884
| align="center" align="center" valign="bottom" | 2.597
| align="center" align="center" valign="bottom" | 96.151
| align="center" align="center" valign="bottom" | 96.169
|- style="background-color:#D9D9D9;font-size:12pt"
| height="15"  valign="bottom" | Perecent On Target
| align="center"  valign="bottom" | &nbsp;
| align="center" align="center" valign="bottom" | 100
| align="center" align="center" valign="bottom" | 0
| align="center" align="center" valign="bottom" | 66.2
| align="center" align="center" valign="bottom" | 30.1
|}
The results are quite encouraging, as in the lane where only C1-C2 (PCAmp1) was added, only C1-C2 shows up. In the two mixed lanes, however, we get reads from both fragments. Because of this, I am investigating further. The "Flip" samples from Positive Control Amplicon Test 2 test the wrong latch-template combination without the other latch or template (e.g. tests LatchX3 and C1-C2). Therefore, sequencing them will give me a better idea of what is going on. I think that the latch is able to bind even when it is wrong. This is because the 18bp sequences flanking each latch are the same (they are the adapter sequences). If that is the case, I should see sequencing results that match the latch, not the padlock.
One additional note. Extracting only the barcodes is not proof enough that the barcodes are in the right place. So I manually aligned three reads. They are all perfect.
[[Media:MiSeq20160822-Analysis.pdf|Alignment Table]]


[[Category:ComboLock]] [[Category:20160822]] [[Category:20160818]]
[[Category:ComboLock]] [[Category:20160822]] [[Category:20160818]]

Latest revision as of 20:37, 26 August 2016

Positive Control Amplicon Test 2 (Started Monday)[edit]

Back to Calendar

I'm going to run library prep on the product from qPCR from Tuesday. I'll take the two "flip" lanes (wrong latch for the amplicon template) because I want to see what is going on. I'll also take the best results from X2 and X3.

Protocol-Library Prep[edit]

  1. Size Selection
    1. Add 20 uL sample lanes 1 and 8-10, 5 uL 6X loading dye and 5 uL TBE to sample lanes
      1. Lane 1: X2-1X
      2. Lane 8: X3-50X
      3. Lane 9: X2-Flip
      4. Lane 10:X3-Flip
    2. Aliquot 30 uL each into 4 sample lanes
    3. 2 uL ladder with 5 uL gel loading dye and 28 uL TBE buffer
    4. Run gel for 20 min at 250V
    5. Stain with 3 uL SYBR Gold for 3 min
    6. Image in gel doc; cut out selected bands and place in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
    7. Centrifuge gel at 14000rpm for 1.5 minutes
    8. Remove 0.5 mL tube and add 500 uL TE buffer to shredded gel
    9. Incubate at 37C for at least an hour with vigorous shaking
    10. Centrifuge at 14000rpm for 1.5 minutes
    11. Transfer supernatant to a nanosep column
    12. Centrifuge at 14000rpm for 1.5 minutes
    13. Transfer flow through to a 2 mL tube
  2. Ethanol Precipitation
    1. Add 1.25 mL 100% EtOH, 50 uL 3M NaOAc, and 2 uL glycoblue to sample
    2. Incubate overnight at -80C; continued tomorrow

Positive Control Amplicon Test (Started 8/18/2016)[edit]

Preliminary Sequencing Results[edit]

From looking at the results from the four sample index lanes. Each of the indexes corresponds to an experiment, I've attached the expected table below.

Sample No Sample Name Index Expected Product
1 Amp1 Test 10 Amp1
2 Amp3 Test 11 Amp3
3 LatchX2 Specificity 12 Amp1
4 LatchX3 Specificity 13 Amp3

Also below is the gel image from size selection. Note that index 11 didn't have any product of the right size, so I don't expect any results from that column.

Now we can look at the results. In order to get these I used grep on the barcodes that are specific to each of the three positive control pieces (C1, C2 and C3). I grepped each barcode alone and in pairs with each other. Note that C1-C2 and C3-C2 are the expected products, C1-C3 should never exist.

Name Barcode Sequence Index 10 Index 11 Index 12 Index 13
Latch6 BC (C3) GTTGGCAG 4 95 7658 11266
Latch8 BC RC (C1) TACAAATA 1140 187 14963 26222
Latch9 BC RC (C2) GTAGGCGC 1069 77 22059 37200
Intersect L8-L9 (C1-C2) TACAAATA,GTAGGCGC 1025 0 14047 25016
Intersect (C3-C2) GTTGGCAG,GTAGGCGC 0 2 7163 10759
Intersect (C1-C3) GTTGGCAG,TACAAATA 0 0 0 0
Percent C2 Reads from Amplicons   95.884 2.597 96.151 96.169
Perecent On Target   100 0 66.2 30.1

The results are quite encouraging, as in the lane where only C1-C2 (PCAmp1) was added, only C1-C2 shows up. In the two mixed lanes, however, we get reads from both fragments. Because of this, I am investigating further. The "Flip" samples from Positive Control Amplicon Test 2 test the wrong latch-template combination without the other latch or template (e.g. tests LatchX3 and C1-C2). Therefore, sequencing them will give me a better idea of what is going on. I think that the latch is able to bind even when it is wrong. This is because the 18bp sequences flanking each latch are the same (they are the adapter sequences). If that is the case, I should see sequencing results that match the latch, not the padlock.

One additional note. Extracting only the barcodes is not proof enough that the barcodes are in the right place. So I manually aligned three reads. They are all perfect.

Alignment Table