Phillip J. Buckhaults, Ph.D. Profile picture
Apr 22 25 tweets 6 min read Read on X
Plasmid DNA copy number quantification. protocols, reagents, and a thread of new results.
this is a map of the plasmid we sequenced from 2020 batches of mRNA vaccine. Image
we made a bunch of primer/probe pairs at IDT. here are the ones that worked the best.




Neo1 FWD

Neo1 REV

Neo1 PRB

Neo5 FWD


Neo5 PRB

pBR322-5 FWD

pBR322-5 REV

pBR322-5 PRB

Spike1 FWD

Spike1 REV

Spike1 PRB

Spike N1 FWD

Spike N1 REV

Spike N1 PRB
we made a synthetic gBLOCK standard and ordered it from IDT. this is the quantification standard on which all our qPCR results rest. so it was critical to have a high-confidence quantitative standard. i trust the number IDT prints on the side of the tube for the quantity of gBLOCK delivered.Image
here is the sequence of the positive control gBLOCK in case you want to order it yourself. it is a frankenstein assembly of a bunch of potential target regions, with signature sequences added so that if we ever sequence we can easily tell the difference between authentic targets coming from the plasmid vs contamination with our positive control. we have no such contamination yet but thought ahead and made it so we could tell the difference (this is for people who carefully analyze the sequence below. you can just ignore all this and simply order as-is and use it.

Here is the recipe for the PCR mix. we use Perfecta toughmix UNG from Quantabio, but probably any PCR reagent for Taqman will work. Image
we checked over a dozen primer pairs and did touchdown gradients to find a PCR condition that worked well for all primer pairs and gave zero background. here it is. touchdown-PCR is one of our favorite ways to make very sensitive (single molecule) but very specific PCR. Image
now for some results from the best primer/probe combinations. Image
summary. Pfizer got cleaner. as i predicted. Image
everyone wants to know ng/dose. this is an underestimate because i am converting copy number to ng assuming no fragmentation (which we know is not true).. most targets will in fact be cut and therefore invisible, but for now the tallest bar is a lower estimate. Image
yes, there was more than 10 ng / dose. i am sure of it now. Image
old pfizer had more than the regulatory limit of 10ng of dna per dose.

new pfizer is cleaner than old pfizer.

new moderna is twenty thousand times cleaner than old pfizer. Image
anything worth doing is worth overdoing.

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More from @P_J_Buckhaults

Feb 28
DNA methylation in NORMAL colon can identify who does and does not have colon cancer. its not perfect, but its pretty good. the loci that are differentially methylated between people with and without cancer are the same loci that change dramatically over time as people age. Cancer patients have normal colons that aged too quickly.
the developmental biologist will know why this is super interesting. HOX genes have the methylation age clock screwed up. Image
pretty wild. Image
Read 6 tweets
Feb 1
Friends, put this on blast worldwide.

I will check for DNA integration. Send me de-identified samples and I will PCR test them and sequence any positives.

You and your institution can remain anonymous and avoid the drama. promise.

@DrJBhattacharya @FLSurgeonGen @TheChiefNerd @drdrew

one useful type of sample would be archived tumor samples from cancers developing six months or more after exposure.
cardiac pathology samples from autopsies of heart attacks occurring less than one month after exposure.
Read 10 tweets
Aug 16, 2023
An open appeal to health care workers around the world. The Pfizer vaccine is contaminated with plasmid DNA. I want to test tissue and blood samples from people recently vaccinated and see if there is any evidence of this DNA integrating into host genomic DNA. PM me if you would like to help. Thanks. -Dr B.
but I think that today because of what people are worried about if I checked 20 or 30 cases of myocarditis, and they were all negative it would put some concerns to rest. And that would be a good thing. Alternatively, if I find it in the myocardium, or the pericardium we have an evidence of an easy fix to a serious problem
@KnowledgeAcqui1 @nellage but even PBMC’s from recently VAX people could be very useful, even if the results are negative. It could eliminate some possibilities.
Read 6 tweets
Aug 15, 2023
@Kevin_McKernan Image
@Kevin_McKernan Image
@Kevin_McKernan Image
Read 9 tweets
Jul 27, 2023
Pfizer vaccine contains pieces of the DNA plasmid that was used for the in-vitro transcription reaction to make the mRNA. I checked two lots by PCR and sequenced by Oxford Nanopore and verified the findings of @Kevin_McKernan. This does not mean there is a problem, however it……
@JohnGal52793009 @Kevin_McKernan but i looked for human dna just for fun
@JohnGal52793009 @Kevin_McKernan so we might be off by twofold in One Direction or another, but we’re not off by tenfold
Read 11 tweets
Jun 29, 2022
Raindrops on roses and whiskers on kittens. Bright copper kettles and warm woolen mittens.
Brown paper packages tied up with string. These are a few of my favorite things.
Cream colored ponies and crisp apple strudel. Doorbells and sleigh bells and schnitzel with noodles.
Wild geese that fly with the moon on their wings. These are a few of my favorite things.
Girls in white dresses with blue satin sashes. Snowflakes that stay on my nose and eyelashes.
Silver white winters that melt into springs. These are a few of my favorite things.
Read 10 tweets

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