Tag Archives: colony screen

PCR – COX/PGS Cloning Colony Screens from yesterday

Performed PCR on 40 colonies using both qPCR primer sets to see if I could differentiate between which colonies potentially contained each isoform to reduce the amount of clones needed for sequencing. Master mix and cycling params are here. Primers used were:

Cg_COX1/2_qPCR_F (SR ID: 1192)

Cg_COX1_qPCR_R (SR ID: 1191)

Cg_COX2_454align1_R (SR ID: 1190)

Positive controls for both primers set were also run. The positive control template was the purified PCR product from 20111006.

Results:

Ladder is Hyperladder II (Bioline). Samples are loaded, left to right, as PGS1 and PGS2 on each colony (e.g. on the bottom gel image, under the “Colony 40″ label is the PGS1 rxn on the left and the PGS2 rxn on the right).

Nearly every colony exhibits amplification using both primer sets, w/the PGS1 reaction producing a band of ~250bp and the PGS2 reaction producing a band of ~750bp. Colonies 18 and 28 are an exception to this and produced no band with the PGS2 primer set. NTCs were clean. The positive controls worked as expected, yielding a band of ~250bp for PGS1 and a band of ~250bp for PGS2.

It is confusing as to why the size of the PGS2 positive control is different than the product that was generated from the colony PCRs.

Will select 10 colonies for mini-preps.

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Colony PCRs – C.gigas COX2/PGS2 Clones (from yesterday)

Performed colony PCRs on the 4 sets of cloning reactions that were performed yesterday using the M13F/R vector primers. Colonies were picked, restreaked on a fresh LB Kan50 plates (made 20110726 by SJW) and PCR’d. Master mix calcs are here. Selected 8 white colonies from each cloning reaction for PCR. Restreaked plate was incubated @ 37C O/N.

Cycling Params:

  • 95C – 10m

40cycles of:

  • 95C – 10s
  • 55C – 10s
  • 72C – 3m

Results:

Hyperladder I is used as the ladder in both gels.

Cloning results look great (except Colony #1 in the 5′ Top Band didn’t produce a product). Will select a re-streaked colony from each set and inoculate liquid culture for mini prep and subsequent sequencing.

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PCR – Colony PCR on Restreaked PGS2 Clones from 20110707

Ran a colony PCR at the same time that I inoculated liquid cultures, using M13 primers.

Cycling params:

  • 94C – 10m

40 cycles of:

  • 94C – 1m
  • 50C – 1m
  • 72C – 2m

Results:

Lane 1: Hyperladder I

Lane 2: PGS Lo 1

Lane 3: PGS Hi 3

Lane 4: PGS Hi 4

Lane 5: Neg. Control

The only colony with an insert is PGS Hi 4. Will run a plasmid prep. However, this is the same sample that was sent for sequencing that produced nothing but vector sequence…

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Colony PCR – Colonies from COX1 Genomic Cloning (from 20110411)

Ran colony PCR on various colonies produced from cloning on 20110411. All colonies were picked, re-streaked on Kan50 plate(s) and PCR’d. Master mix calcs are here. Cycling params:

  • 95C – 10mins

40cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 3mins
  • 72C – 10mins

Results:

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Colony PCR – 5’RACE Colony: COX2 (repeat of yesterday’s PCR)

Repeated yesterday’s PCR on the re-streaked colony in order to run the product on a gel with a more appropriate ladder. See yesterday’s entry for all PCR info.

Results:

Lane 1: Hyperladder I

Lane 2: colony PCR

Lane 3: NTC

A band of nearly ~950bp is seen in the colony PCR, suggesting that the cloning reaction was successful. However, this does not match up with the expected size of ~1500bp seen on 20110407. Will sequence this regardless. Also, the gel on 20110407 may not have run properly (see image from that dat), which could possibly explain why we don’t see the “expected” size band of ~1500bp? Will inoculate a liquid culture for mini prep for eventual sequencing.

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Colony PCR – 5′ RACE Colony: COX2

One white colony (marked with arrow in image linked) from the two plates from Steven’s cloning (from yesterday) was picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix – 25uL

10uM M13 Forward – 1uL

10uM M13 Reverse – 1uL

H2O – 23

Added 25uL to each PCR tube.

Cycling Params:

  • 95C – 10mins

40 cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 2mins

1 cycle:

  • 72C – 10mins

Results:

Lane 1: Hyperladder IV

Lane 2: colony PCR

Lane 3: NTC

Turns out the Hyperladder IV (this gel was run in the Friedman Lab) only goes up to 1000bp. So, the band we see in the colony PCR reaction could be close to the expected size if the insert is present (~1500bp). Although, we also see a band in the NTC. Will repeat this PCR and run on a gel with a more appropriate ladder…

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Colony PCR – 5′ RACE Colonies

Two light blue colonies (there were no white colonies) were picked, restreaked on a new Kan50 plate (no X-gal) and PCR’d.

Master Mix:

2x Apex Red Master Mix – 37.5uL

10uM M13 Forward – 3uL

10uM M13 Reverse – 3uL

H2O – 46.5

Added 25uL to each PCR tube.

Cycling Params:

  • 95C – 10mins

40 cycles of:

  • 95C – 30s
  • 55C – 30s
  • 72C – 2mins

1 cycle:

  • 72C – 10mins

Results:

The two pale blue colonies do NOT contain our desired insert. Despite the presence of a larger, faint band (~950bp), that is far smaller than our 5’RACE insert size (~1500bp). And, clearly, the primary amplicon produced is ~250bp, which is the expected size for empty vector… Ladder is Hyperladder I (Bioline).

Will need to re-do ligation reaction and will do so at the recommended volumes in the TOP TA (Invitrogen) protocol.

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