The 1989 Molecular Cloning Manual: Why Labs Still Cite It

Where Did the Molecular Cloning Manual Come From?

The manual started as course material. The book grew out of protocols used on a gene-cloning course run at Cold Spring Harbor Laboratory in summer 1980, taught by Tom Maniatis, Ed Fritsch and Nancy Hopkins (Creager, 2020). Maniatis, Fritsch and Joe Sambrook published it as Molecular Cloning: A Laboratory Manual in 1982 (Creager, 2020).

Demand was immediate. Advance orders passed 5,000 copies, and Cold Spring Harbor Laboratory Press sold 5,113 copies in July 1982, its first month on sale (Creager, 2020). In 1983 the press sold more copies of this one title than of all its other books combined: 37,337 against 24,234 (Creager, 2020).

A bench book with nicknames

Bench users in the 1980s called the book “Maniatis”, after its first author, and many referred to it simply as the bible (Creager, 2020). One Berkeley user remembered the “spiral-bound Maniatis” (Creager, 2020).

The historian Angela Creager of Princeton titled her 2020 history of the book “Recipes for recombining DNA”, and the label fits (Creager, 2020). The manual reports no experiments of its own. It gathers step-by-step laboratory procedures centred on nucleic acids (Creager, 2020; Dunn et al., 2019).

Counting later editions, the manual sold more than 200,000 copies (Creager, 2020). Sambrook stayed with it through four editions until his death in 2019 (Dunn et al., 2019).

What Changed in the 1989 Second Edition?

The second edition was larger, later than planned and signed in a new order. A revision scheduled for 1984 was deferred for five more years while the first edition went through sixteen printings (Creager, 2020). Creager reports that the rapid pace of change was blamed for the delay, with the polymerase chain reaction, which she dates to 1985, the most prominent example (Creager, 2020). She quotes David Crotty of Cold Spring Harbor Laboratory Press, who argued that after PCR a new edition without it would be “pointless” (Creager, 2020).

When it did appear in 1989, the new edition filled three thick volumes and kept the plastic ring-comb binding of the first (Creager, 2020). The author order was reversed to Sambrook, Fritsch and Maniatis (Sambrook et al., 1989; Creager, 2020). That is why the same manual turns up in reference lists both as Maniatis et al. 1982 and as Sambrook et al. 1989. Sambrook, now first author, would steer the book through two more revisions (Dunn et al., 2019).

Two editions since

Sambrook and David Russell wrote the third edition, dated 2001, which came with a CD-ROM and the MolecularCloning.com website (Creager, 2020). Michael Green and Sambrook wrote the fourth, published in 2012 as a three-volume paperback of 2,028 pages (Green & Sambrook, 2012). Its publisher describes 12 entirely new chapters, among them epigenetic analysis, RNA interference, genome sequencing and bioinformatics, plus 10 updated core chapters (Green & Sambrook, 2012). In September 2026 the publisher still listed the fourth edition, and we found no fifth (Green & Sambrook, 2012).

Table 1. The four editions of Molecular Cloning: A Laboratory Manual

How Often Has the Manual Been Cited?

Molecular Cloning ranks among the most-cited works in science, and Nature described it as a mainstay of molecular biology laboratories (Van Noorden et al., 2014). In 2014 Nature compiled the 100 most-cited papers from Thomson Reuters’ Web of Science and set beside them a list from Google Scholar (Van Noorden et al., 2014). Two-thirds of the Google Scholar list were books, which Thomson Reuters did not analyse, and there the manual came fourth, the most-cited book on the list (Van Noorden et al., 2014). Because the Thomson Reuters ranking left books out, the manual is absent from it (Van Noorden et al., 2014).

Google Scholar counted the editions separately: 172,540 citations for the 1989 edition and 61,929 for the 1982 edition (Van Noorden et al., 2014). Nature noted that it could not match every Google Scholar item exactly and printed the data as received (Van Noorden et al., 2014). Both editions are flagged in its table as books not included in Web of Science (Van Noorden et al., 2014).

Creager’s own search of Web of Science shows how quickly the habit formed. More than 7,000 articles cited the manual in 1989 alone (Creager, 2020). From 1991 to 2000, editions of Molecular Cloning drew over 10,000 citations a year, while the rival Current Protocols in Molecular Biology peaked at 2,520 citations in 1998 (Creager, 2020).

Table 2. Citation figures for Molecular Cloning from two published analyses

These numbers are snapshots from 2014 and earlier, but they show the scale: for decades, citing Sambrook or Maniatis was a routine part of writing up molecular biology methods (Creager, 2020; Van Noorden et al., 2014).

Why Do Methods Sections Still Cite It?

A reference to Sambrook et al. 1989 almost always names a procedure, not a result. Because the manual reports no findings of its own, authors cite it to show where a standard technique came from (Sambrook et al., 1989; Creager, 2020). Both pages linking to this archive record that we opened use it in that way (Cavalcante et al., 2020; Hameed, 2024).

The first is a 2020 BMC Medical Genetics study of insertion–deletion (INDEL) variants in apoptosis genes and gastric cancer (Cavalcante et al., 2020). Its methods say DNA was extracted with the phenol-chloroform method, based on the manual, and the reference points to the Cabdirect record (Cavalcante et al., 2020). Nothing in the paper’s findings depends on the manual beyond that step.

The second is a 2024 paper in the Wasit Journal for Pure Sciences on oral bacteria and biofilm formation (Hameed, 2024). It cites the manual for keeping Streptococcus cultures at −20 °C in brain-heart infusion broth containing 20% glycerol (Hameed, 2024). The next sentence says the method kept cultures for up to three months, but that claim is the author’s own (Hameed, 2024).

Phenol-chloroform extraction in the current protocols

The organic-solvent method the BMC paper used has not disappeared. In a 2017 Cold Spring Harbor Protocols article on high-molecular-weight DNA, Green and Sambrook describe protein removal as the key step, often achieved by extracting nucleic acid solutions with phenol:chloroform and then chloroform (Green & Sambrook, 2017). A lab can therefore cite a current, peer-reviewed version of the recipe rather than the 1989 volumes (Green & Sambrook, 2012; Green & Sambrook, 2017).

A citation to Sambrook et al. 1989 tells a reader which recipe a lab started from, not what it actually did at the bench.

An old citation also hides detail. Wording such as “based on” leaves open what a lab changed (Cavalcante et al., 2020), and readers without the 1989 volumes cannot easily check. Methods sections that name the edition and the exact protocol are easier to repeat.

How Have Routine Bench Methods Changed Since 1989?

Two clear shifts came in DNA purification and in the chemicals around the gel tank. A year after the second edition, Boom and colleagues described a silica and guanidinium method that purified nucleic acids from 12 specimens in under an hour (Boom et al., 1990). The DNA was significantly sheared but usable, and yields were usually over 50% (Boom et al., 1990).

From organic solvents to kits

A 2009 review traces how extraction moved on from laborious, low-throughput procedures (Tan & Yiap, 2009). Researchers can now choose among solution-based and column-based kits, and demand for automated systems has grown (Tan & Yiap, 2009). Phenol-chloroform extraction is still described in current protocols, but it is no longer the only standard route (Green & Sambrook, 2017; Tan & Yiap, 2009).

Gel stains

Ethidium bromide, the classic stain for DNA in agarose gels, has a direct link to the manual’s authors. Sambrook’s obituarists credit him with first developing its use on gels of DNA fragments cut by restriction enzymes (Dunn et al., 2019).

In a 1999 Ames test, ethidium bromide raised revertant counts in frameshift strains of Salmonella about 68-fold over controls in strain TA98, but only with a mammalian microsome (S9) mix added (Singer et al., 1999). SYBR Green I was a weak mutagen by comparison (Singer et al., 1999). These are bacterial results, not measures of human risk, and the lead author worked at Molecular Probes, the stain’s maker (Singer et al., 1999).

How Is DNA Cloned Today?

Cloning today can draw on one-tube methods published about two decades after the 1989 edition (Gibson et al., 2009; Engler et al., 2008). Gibson assembly joins several overlapping DNA pieces in a single reaction, while Golden Gate cloning moves a fragment into a vector in one step (Gibson et al., 2009; Engler et al., 2008). Golden Gate extends the older cut-and-ligate approach rather than abandoning it, since it still relies on restriction enzymes and ligation (Engler et al., 2008).

Gibson assembly and Golden Gate

Daniel Gibson and colleagues published the method in 2009, and the whole reaction runs at one constant temperature (Gibson et al., 2009). Three enzymes work together in it: a 5′ exonuclease, a DNA polymerase and a DNA ligase (Gibson et al., 2009).

Golden Gate cloning, published in 2008, uses type IIS restriction enzymes (Engler et al., 2008). Its authors reported close to 100% correct recombinant plasmids after a restriction-ligation lasting just five minutes (Engler et al., 2008).

From printed volumes to online protocols

The manual itself adapted. Its fourth edition recast the content around nucleic acid methods and added chapters on genome sequencing and bioinformatics (Green & Sambrook, 2012). In March 2006 the publisher announced CSH Protocols, an online database of step-by-step methods in the style of its manuals, built with Stanford’s HighWire Press and sold by site licence (Cold Spring Harbor Laboratory, 2006). Release was planned for June 2006 (Cold Spring Harbor Laboratory, 2006).

Protocols by Green and Sambrook, such as a 2017 method for high-molecular-weight DNA, now appear in Cold Spring Harbor Protocols (Green & Sambrook, 2017). For a reader who meets Sambrook et al. 1989 in a paper today, the reference marks a starting recipe, and the current versions sit in the fourth edition and in the journal (Green & Sambrook, 2012).

Frequently asked questions

Adrian Foster — author

Adrian Foster writes with an editorial focus on experimental science, laboratory research, controlled comparisons, measurement techniques and the role of replication in evaluating scientific findings. At CAB Direct, Adrian covers experiments, laboratory method...