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clarifications to protocols

It had been a while since I looked at the bench-ready protocols we have online. I've updated them with some minor changes based on improved lab decontamination strategies, and made little changes to hopefully make some parts of the protocols a little more clear.
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alliblk committed Aug 17, 2018
1 parent 345f476 commit 246ec0b8e7551f12dc83ce1eecfdc309192362ed
@@ -1,6 +1,6 @@
# Two-step RT-PCR for amplicon generation

_Perform the following in a hood or in a pre-PCR designated area_
_Perform the following in a clean pre-PCR hood or in a pre-PCR designated area. The hood and all pipettes/tips entering the hood should be cleaned with 10% bleach and 70% ethanol prior to use._

#### Reverse Transcription

@@ -16,9 +16,11 @@ _Perform the following in a hood or in a pre-PCR designated area_
* 10 uL ProtoScript II Reaction Mix
* 2 uL ProtoScript II Enzyme Mix

_Note: you can also make the this as a mastermix, and pipette once per sample._

5. Place on the thermocycler and run the following program:

* 25 degrees Celsius for 5 minutes _**note: this step can be swapped for a 5 minute hold at room temperature_
* 25 degrees Celsius for 5 minutes _**note: this step can be swapped for a 5 minute hold at room temperature._
* 48 degrees Celsius for 15 minutes
* 80 degrees Celsius for 5 minutes
* Hold at 10 degrees Celsius
@@ -45,4 +47,4 @@ _Perform the following in a hood or in a pre-PCR designated area_
* 65 degrees Celsius for 5 minutes
* Step 3: Hold at 4 degrees Celsius.

_**note: If generating amplicons for Miseq run rather than MinION run, you should do only 35 cycles of amplification, since you will have additional cycles of amplification during library prep_
_**note: If generating amplicons for Miseq run rather than MinION run, you should do only 35 cycles of amplification, since you will have additional cycles of amplification during library prep._
@@ -4,19 +4,19 @@

_**update: from experience I've found that a 1x bead clean-up is preferable to a 1.8x clean-up if you are sequencing on the MinION. The MinION library prep features 1x clean-ups throughout the protocol, and I've found that if a 1.8x clean-up is used on the amplicons it's hard to keep the library concentration high enough throughout the prep, probably because the 1x clean-ups are more stringent bottlenecks. If you're going to be sequencing on the MiSeq, it should be fine to stick with the 1.8x clean-up._

1. Label two sets of DNA Lo-Bind tubes (one set for bead clean-up and the other for eluate with cleaned up amplicons)
2. Allow AMPure XP beads to come up to room temperature, and homogenize by vortexing.
3. Add 40 or 72 uL of AMPure XP beads to each tube in one set of the Lo-Bind tubes depending on sequencing platform.
4. Pipette 40 uL of PCR product into correspond tube with beads.
5. Incubate beads and PCR products at room temperature on a hula mixer for 5 minutes. _if no access to a hula mixer, you can flick mix gently throughout the incubation_
1. Label two sets of DNA Lo-Bind tubes (one set for bead clean-up and the other for eluate with cleaned up amplicons).
2. Allow AMPure XP beads to come up to room temperature, and homogenize thoroughly by vortexing.
3. Add 40 uL (1x) or 72 (1.8x) uL of AMPure XP beads to each tube in one set of the Lo-Bind tubes depending on sequencing platform.
4. Pipette 40 uL of PCR product into corresponding tube with beads.
5. Incubate beads and PCR products at room temperature on a hula mixer for 5 minutes. _if no access to a hula mixer, you can flick mix gently throughout the incubation period._
6. Place tubes on magnetic rack and incubate until the solution is clear.
7. Discard the supernatant being careful to not disturb pellet.
8. Add 200 uL of 80% EtOH to each tube to wash pellet, incubate 30 seconds, then discard EtOH wash.
8. Add 200 uL of 80% EtOH to each tube to wash pellet (while being careful to not disturb pellet), incubate 30 seconds, then discard EtOH wash.
9. Repeat previous wash again, pipetting off as much EtOH as possible.
10. Spin tubes down, replace on magnetic rack, and pipette off any additional EtOH. Leave tubes open to dry.
11. Allow pellets to dry (roughly 5 minutes). The pellet should appear matte but not dry to the point where cracks form.
11. Allow pellets to dry (roughly 5 minutes). The pellet should appear matte but not dry to the point where cracks form. DNA yield will drop if the pellet is left to dry too long.
12. When pellet is dry add 31 uL of nuclease-free water to each tube, remove from rack and flick gently to resuspend beads. _if storing amplicons for longer periods of time, qiagen Elution Buffer can be used instead of nuclease-free water._
13. Once pellets have been resuspended incubate at room temperature on a hula mixer for 5 minutes.
13. Once pellets have been resuspended, incubate at room temperature on a hula mixer for 5 minutes.
14. Replace tubes on magnetic rack and incubate until solution fully clears.
15. Carefully pipette off 31 uL of supernatant without disturbing beads and place into new Lo-Bind tubes.

@@ -35,6 +35,6 @@ _You should use the Qubit High Sensitivity dsDNA kit_
5. Vortex, spin down and wait 2 minutes.
6. Quantify the concentration of dsDNA in the sample.

* Negative extraction controls usually have concentrations around 3 or 4 ng/uL.
* Negative PCR controls should have concentrations < 1 ng/uL.
* Negative extraction controls usually have concentrations around 3 or 4 ng/uL. If you have access to gel electrophoresis, you can run these negatives on a gel to check whether there is amplicon contamination or whether the quantifiable levels of dsDNA are simply represent a smear.
* Negative PCR controls should have concentrations < 1 ng/uL. Ideally these are unquantifiable, but it is challenging to get concentrations this low except in completely ideal lab settings.
* Properly amplified samples should have concentrations between 5 and 100 ng/uL.
@@ -8,6 +8,8 @@ _Other equivalent extraction methods can also be used (e.g. Trizol, Omega etc.)_

_In addition to the samples, remember to have an extraction negative control (add nuclease-free water instead of serum/urine)._

_We recommend that this protocol be carried out in a clean, pre-PCR hood that has been wiped down with bleach and ethanol before use. If you have particular concern about sample cross-contamination, gloves should be wiped with a bleach-soaked towellete between each sample. Until particle lysis is complete (end of incubation in step 6), there are infectious virions in the sample. All pipette tips and tubes should be discarded and soaked in 10% bleach for 1 hour to sterilize._

1. Remove serum samples from freezer and allow to come up to room temperature.
2. Prepare stock solution of Buffer AVL + carrier RNA. _Per sample_ add:

@@ -34,4 +36,4 @@ _In addition to the samples, remember to have an extraction negative control (ad
17. Place column in clean, labelled 1.5 mL tube. Add 50 uL of Buffer AVE at room temperature to column.
18. Incubate at room temp for 1 minute.
19. Centrifuge at room temp at 8000 rpm for 1 minute.
20. Place extracted RNA in eluate on ice if proceeding directly to PCR or store in freezer.
20. Place extracted RNA on ice if proceeding directly to PCR or store at -80 degrees Celsius.

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