CHC extraction vials

Lab protocols

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Lab protocols

Enter the name for this tabbed section: DNA pico green quantification
PicoGreen quantification 

.pdf version

# PicoGreen quantification #

The PicoGreen kit (dye, DNA standard and buffer catalogue number [P7589]( cost £320 on 26/01/2012.

It is sensitive enough to detect 50 pg of DNA regardless of assay volume (200 ul for plate or 2 ml for cuvette).

To make the standard, we can prepare the desired concentrations in separate eppendorfs and dilute them 100-fold for the final assay. We can input their original dilution in the software to calculate concentrations so that our samples that are diluted equally are quantified without conversion. For the plate, the minimum possible concentration that is quantified is 50 pg / 200 ul = 0.25 pg/ul original concentration cannot be accurately quantified with a 100-fold dilution.

# Procedure #

Make a 400-fold dilution of the PicoGreen in TE that was used for the DNA.

* For one plate (100 samples x 200 ul per well) use 19.950 ml TE and 50 ul of PicoGreen.

* For two plates, 39.9 ml TE and 100 ul PicoGreen.

Pipette 2 ul of all DNA samples to be quantified on each well of the plate.

Pipette 2 ul of all ladder standards, except for the 100 standard (4 ul) and the 6.25 standard (1 ul). Leave one well empty as a 0 point for the ladder.

Add 198 ul recently made PicoGreen solution to each well.

Ready for the plate reader.

# Preliminaries #

## Assay Buffer ##

Dilute the 20X TE buffer with DNase-free water. It is 25 ml so you can add 475 ml DNase-free water.

## Picogreen ##

The manual makes a 200-fold dilution of PicoGreen to prepare a working solution, and uses it in a 1:1 ratio with the samples to be quantified, so the final PicoGreen dilution is 400-fold.

## DNA Standard ##

The lambda DNA standard is at 100 ug / ml

To make standards:

**100 ug/ml**: pipette 100 ul original standard into a new eppendorf
**50 ug/ml**: pipette 50 ul of above and 50 ul TE into new eppendorf
**25 ug/ml**: pipette 50 ul of above and 50 ul TE into new eppendorf
**12.5 ug/ml**: pipette 50 ul of above and 50 ul TE into new eppendorf
**6.25 ug/ml**: pipette 50 ul of above and 50 ul TE into new eppendorf

Enter the name for this tabbed section: QIAGEN RNA extraction

.pdf version

# QIAGEN RNA extraction from parts of *Drosophila* samples stored in RNAlater

RNAlater is meant for animal organs, such as kidney. The crystals are supposed to be removed before using the tissue. For small samples, such as *Drosophila* testes or accessory glands, the salts are removed by adding an equal volume of PBS to dissolve the salts. Most are then removed after spinning the samples, by pipetting off the supernatant.

Dissections of *Drosophila* are best done on an ice block covered with a black rubbish bag, in RNAlater. The dissected tissues are then moved to 200 ul RNAlater per sample.

This protocol produces close to 1 mg clean total RNA from 100 *Drosophila* female reproductive tracts (excluding ovaries).

## Before Starting ##

* Make sure RPE has had ethanol added.
* Add 550 ul RNase free H2O to the glass vial using a syringe.
* Invert the vial to mix the contents
* Remove the foil cover, open then lid and pipette off the solution to aliquots to store at -20oC, e.g. 40 ul for 4 samples at a time.
* Add 10 ul beta-mercaptoethanol (dangerous) per 1 ml RLT in a fume hood.
* 14 ul in 1400 ul RLT for 4 samples
* Prepare fresh 70% ethanol
* 420 ul RNase free H2O and 980 ul Ethanol.
* Label new tubes and add 350 ul of freshly made 70% EtOH in each tube.

## Procedure ##

Assumes 4 samples are done at a time, and they started in 200 ul RNAlater.

1. Add equal to RNAlater volume (200 ul) PBS to RNAlater samples, invert to mix and dissolve crystals, spin for 10 min to pellet the tissues.
2. Remove as much of the supernatant as possible.
3. Add 200 ul RLT.
4. Homogenise with spinning machine. White tips work best.
5. Add 150 ul RLT to each vial.
6. Centrifuge for 3 min.
7. Transfer supernatant to already labelled tube with 600 ul 70% EtOH, mix by pipetting,
8. Transfer 700 ul at a time, along with precipitate, to RNeasy spin column inside a 2ml tube. Close lid and spin for 15s at > 10,000 rpm.
9. Discard flow-through. Repeat until all sample is used.
10. (Optional) on-column DNA digestion happens here. If so
1. Add 350 ul RW1, close lid and roll/invert tube, spin for 15 sec, discard flow though.
2. DNase digestion
1. Make DNase from pre made frozen eppendorf with 40 ul DNase mixed with 320 ul buffer RDD.
2. Pipette 80 ul DNase mix to the each spin column membrane
3. Incubate for 15 min at room temperature.
3. Add 350 ul RW1, close lid and roll/invert tube, spin for 15 sec, discard flow though.
11. (only if did not do DNase) Add 700 ul RW1. Spin for 15 sec at 10,000 rpm. Discard flow-through.
12. Add 500 ul RPE, close lid and roll/invert tube, spin for 15 sec, discard flow though.
13. Add 500 ul RPE, close lid and roll/invert tube. Spin for **2 min** at 10,000 rpm.
14. Place RNeasy column in new 2ml collection tube and spin for 5 min with open lids to completely dry membrane.
15. Place spin column in 1.5 ml collection tube. Add 30-50 ul RNase-free water on column membrane. Spin for 1 min to elute RNA.
16. (Optional) Repeat to increase yield.

Enter the name for this tabbed section: NH4Ac DNA extraction

.pdf version

# NH4Ac DNA extraction

1. Make sure there is ethanol in the freezer.
2. Place in a 1.5ml eppendorf tube 200 ul Digsol buffer and 10 ul Proteinase K (10mg/ml).
3. Remove fly from ethanol (with forceps) and blot onto tissue. When dry, transfer to the eppendorf.
4. Disrupt the fly with electric pestle machine.
5. Wrap the eppendorf rack in tissue paper and elastic bands and place in rotating oven at 55oC (3hrs) or 50oC (overnight).
6. (Optional) Add 3 ul 10ug/ul RNase A[^rnaseNote] and incubate at 37oC for 45 min.
7. Add 300 ul 4M ammonium acetate to each sample.
8. Vortex several times over a period of at least 15 minutes at room temperature to precipitate the proteins.
9. Centrifuge[^centrifugeNote] for 10 minutes at 13,000 rpm [^rpmNote]. Label new eppendorfs.
10. Aspirate 500 ul supernatant into the clean labelled 1.5ml eppendorfs[^pelletnote].
11. Add 1ml 100% ethanol and mix by inverting. The DNA should precipitate out of solution, but may not be visible from single flies, especially males.
12. Leave samples in freezer at -20 oC for 1h.
13. Centrifuge for 10 minutes at 13,000 rpm.
14. Pour off ethanol in a smooth movement so as not to lose DNA pellet.
15. Add 500 ul 70% ethanol and invert several times to rinse pellet.
16. Centrifuge for 5 minutes at 13,000 rpm[^repeat70%Note].
17. Pour off ethanol in a smooth movement and stand tubes upside-down on clean tissue until they are completely dry (1 h or overnight incubation).
18. Once fully dry add Low TE. The amount added is dependent on pellet size, 20 ul results in up to 50 ng/ul DNA from a single female *D. pseudoobscura*.
19. Place tubes in shaking incubator for 30 minutes (55 oC) to dissolve pellet (flicking every 10 mins). Or leave overnight at room temperature. You are aiming for a fully homogeneous, slightly viscous solution.
20. Store at -20 oC (long term) or 4 oC (short term).

# Recipes #

## 1M Tris-Base (MW 121.1) pH 8.0 ##

(Tris Hydroxymethyl Aminomethane)

For 200ml:

* Dissolve 24.22g in distilled water by stirring
* pH should be about 8.0
* Autoclave to sterilise

## 0.5M EDTA (MW 372.2) pH 8.0

(EthyleneDiamineTetraacetic Acid)

For 200ml:

* Dissolve 37.2g in distilled water by stirring.
* Will need to pH solution with NaOH whilst it is dissolving (in order for all EDTA to solubilise).

## 20% SDS

(Sodium Dodecyl Sulphate)

For 100ml:

* Add 20g SDS (use autoclaved water as end solution cannot be autoclaved).
* Use a fume hood and wear a mask when weighing this powder.
* It is easier to dissolve by heating at 55 oC than by shaking.

## Digsol pH 8.0

(Digestion Solution - Bill Amos and Josephine Pemberton)

| Final Concentraction | Stock | For 1000ml | For 200ml |
| -------------------- | ------------------- | ---------- | --------- |
| 20mM EDTA | EDTA (0.5M, pH 8.0) | 40ml | 8ml |
| 120mM NaCl | NaCl | 6.85g | 1.37g
| 50mM Tris | Tris (1M, pH 8.0) | 50ml | 10ml |
| | Distilled water | 810ml | 172ml
| SDS (0.5%) | SDS (20%) | 25ml | 5ml |
[Digsol recipe]

* Warm all constituents except SDS until dissolved
* Autoclave to sterilise
* Add SDS
* pH with HCl if necessary

## 4M Ammonium Acetate, pH 7.5

For 100ml

* Dissolve 30.83g Ammonium acetate[^NH4AcNote] in distilled water.
* Autoclave to sterilise.
* If necessary pH with Glacial acetic acid (was not necessary).

## T.E. pH 7.5-8.0 ##

| Final Concentration | For 400ml |
| --------------------- | ----------- |
| 10mM Tris | 4ml of 1M Tris (pH 8.5) |
| 1mM EDTA | 800 ul of 0.5M EDTA (pH 8.5) |
[TE recipe]

## Low EDTA T.E. Buffer pH 7.5-8.0

For resuspending DNA which will be used in PCR

| Final Concentration | For 400ml |
| --------------------- | ----------- |
| 10mM Tris | 4ml of 1M Tris (pH 8.5) |
| 0.1mM EDTA | 80 ul of 0.5M EDTA (pH 8.5) |
[Low TE recipe]

* pH if necessary
* Autoclave to sterilise

# 10 mg/ml Proteinase K

In 1ml aliquots in -20 oC freezer.

In St Andrews it is kept in special solution at 4 oC at 20 mg/ml.

[^NH4AcNote]: Ammonium acetate is hydrophilic and therefore most of the stock chemical is very wet, however this does not seem to affect the extraction process.

[^repeat70%Note]: If very clean samples are required, repeat the 70% wash steps. However if there will be follow-up cleanup of the samples (like RNase treatment), a single wash is sufficient and reduces the risk of a lower yield.

[^rpmNote]: RPM (revolutions per minute) can be converted to RCF (relative centrifugal force). A common centrifuge gives about 10,000 RCF, I have found that higher RCF (18,000) give better pellets and cleaner supernatants so it is worth finding a more powerful centrifuge is possible.

[^rnaseNote]: Keeping RNA in the samples will make the nano drop quantification unreliable, there can easily be 5-10x more RNA than DNA in the final sample. However for low DNA volumes, having more nucleic acid seems to increase DNA yields because it allows for easier precipitation.

[^centrifugeNote]: Always mix by inverting the eppendorfs while loading to the centrifuge.

[^pelletnote]: Sometimes (depends on temperature and centrifuge) some of the salt comes out of solution and forms part of the pellet (white). Other times it does not, but it forms a thicker layer close to the bottom of the eppendorf. It can be shown to be salt by adding ethanol, and this thick part becomes white. When aspirating the supernatant be very careful not to include the thick bottom layer, which may extend to the sides of the bottom of the eppendorf.

Enter the name for this tabbed section: CHC extraction Drosophila
Enter the name for this tabbed section: Lab notebook
.pdf version

# Lab Notebook suggestions

The notebook should be of the bound hardback type (not a loose leaf file). Entries should be made with black ink or black ballpoint pen (to permit clear photocopying if required). Pencil should not be used (so that there is no suspicion of later changes to data).

The first page should give your personal information.

The next four pages should be reserved for the notebook index which should be filled in as the notebook entries are completed.

The next page should be reserved for abbreviations which will be used in the notebook and it should be filled in as new abbreviations occur during the course of writing up work.

Number all the pages consecutively in the top right corner. It is recommended that as a safeguard in the event of possible legal problems, every page should be used in sequence with no blank pages or spaces remaining. This requires that any blank page or space remaining at the bottom of a page is 'crossed' and signed and dated with the statement 'No entry'. Pages should not be torn out or added as this can lead to doubts about the authenticity of entries. Deletions, corrections or additions should be initialled and dated by the author. If something needs to be added as an afterthought at a later date it should be entered with the date of writing and the statement 'continued from ...' and then cross referenced on the appropriate page with the statement 'related work or notes on pages ... '.

The date on which work is conducted should be entered near the outer margin so that it can then easily be found when quickly checking through for some particular entry - although if the index is kept up to date this should present no problem!

The style used for writing up the notebook will be a personal choice but the aims are clarity, accuracy and completeness. Usually each experiment or set of observations can be divided into

* Background and aims
* Plan
* Description of how work is done
* Record of all results
* Analysis of results
* Conclusion

Using these or similar headings in the notebook will help ensure that all are given adequate coverage. If the plan includes a detailed description of a method, then a note of any specific safety hazards and precautions should be made. When recording results, enter everything directly into the notebook when it happens. Do not keep notes on scraps of paper to write up later - the notes get confused or lost, 'spare' time is always elusive and there is a temptation to leave it until tomorrow with the result it may never be done properly.

The back of the notebook can hold protocols, ladder pictures, sample usage information (for lab bills) etc.
Enter the name for this tabbed section: Qubit
  1. Prepare N tubes + 2 for CS1 and CS2

  2. Prepare master mix (MM)

    N tubes + 2 (CS1, CS2) + 10%

    Solution 1x (ul)
    Buffer 199
    Fluorochrome 1
    Final volume 200

    Vortex well and use within 1 hr.

  3. Prepare the tubes

    CS1: 190 ul (MM) + 10 ul (CS1) CS2: 190 ul (MM) + 10 ul (CS2) Samples: 198 ul (MM) + 2 ul (DNA)

    Vortex well and centrifuge the tubes and let stand for 5 min.

  4. Use Qubit

    Select 'Quant IT dsDNA HS'

    1. Put CS1 and read
    2. Put CS2 and read
    3. Keep CS2 and 'sample' > 'calculate sample concentration' > '10 ul' > 'ng/ul'
    4. Add the first sample 'read' > 'calculate sample concentration' > '2 ul' > 'ng/ul'

    Remember to remove the last stample from the machine.

    Record your usage in the log.