Tag Archives: PCR

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.

5’/3′ RACE PCRs – Nested PCRs for COX2 Sequence

Due to the failure of the primary PCR on both 5′ and 3′ RACE cDNA libraries (from 20080619) yesterday, will perform nested PCR using a nested GSP designed by Steven (CgPGSRACEsrNGSP1; SR ID:1209). The 5uL of PCR reactions that were set aside yesterday were diluted to 250uL with tricine-EDTA (supplied with the Clontech SMARTer RACE cDNA Amplification Kit) as instructed in the Clontech manual. The master mix and tube layouts were exactly the same as yesterday’s, but instead of using 2.5uL of RACE cDNA library as template, I used 5uL of the diluted PCR reaction. Additionally, Universal Nested Primers were used instead of the Universal Primer Mix (both supplied in the kit). Cycling parameters followed “Program 2″ from the Clontech manual for 25 cycles.

Entire PCR rxns were run on a 1.2% gel, as instructed by the Clontech manual.

Results:

So….. What we see here is a melted gel!

But! We also see a successful PCR! The first three lanes (excluding the Hyperladder I) are 5′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). The next three lanes are the 3′ RACE rxn, followed by two different negative controls (the negative control in the last lane is the one we’re really concerned with and it’s totally clean). As hoped/expected, we got a nice, clear product in the 5′ RACE rxn.

The bright band (~1500bp) in the 5′ RACE rxn PCR was excised and purified using Millipore Ultrafree DA columns according to protocol. Will clone and sequence this product.

5’/3′ RACE PCRs – COX2 Sequence on 5′ & 3′ RACE Libraries (from 20080619)

Ran PCRs on both the 5′ & 3′ RACE libraries created 20080619 with a new COX2 gene-specifc (GSP) primer designed by Steven (CgPGSRACEsrGSP1; SR ID: 1208). Although this primer was designed to obtain additional 5′ sequence, it was used with both 5′ and 3′ libraries as a precaution in case it accidentally designed on the wrong strand. PCR rxn was set up according to the Clontech SMARTer RACE cDNA Amplification Kit. Master mix calcs are here. PCR cycling followed “Program 1″ from the Clontech manual for 25 cycles.

After PCR completion, 5uL were transferred to a clean tube and saved, in case this PCR didn’t work and a nested PCR would need to be performed. This is according to the Clontech protocol. Samples were run on a 1.2% agarose gel, as instructed in the Clontech manual.

Results:

No bands of any kind in any sample, including the negative controls (gel not shown). Will perform nested PCR on both libraries in hopes of getting bands.

PCR – New C. gigas COX Primers for Sequencing of Isoforms

Used new primers for obtaining bands for additional sequencing of both COX isoforms in C. gigas. Master mix calcs are here. Master mix shorthand (MM##) is described below:

MM07 – Cg_COX_416_F (SR ID: 1193) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~1540bp

MM08 – Cg_COX_416_F (SR ID: 1193) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~1540bp

MM09 – Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191) Expected band size (if no intron) = ~225bp

MM10 – Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~225bp

MM11 – Cg_COX_1519_F (SR ID: 1146) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~275bp

MM12 – Cg_COX_982_F (SR ID: 1151) + Cg_COX2_454align1_R (SR ID: 1190) Expected band size (if no intron) = ~812bp

Results:

Ladder is Hyperladder I from Bioline.

Master mixes are indicated underneath each group by the labels MM##. The order within each MM group (from left to right) is: template, NTC, NTC.

All bands boxed with green were purified using Millipore’s Ultrafree-DA spin columns. Samples were stored @ -20C in “Sam’s Misc. -20C Box”.

MM07 – Fails to produce any bands of any size. Suggests the presence of intron(s) causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions.

MM08 – Produces a band of ~400bp which is well below the expected 1540bp (if no introns) size. Due to the faintness of the band, the band was not excised. Will consult with Steven to see if he thinks it worth repeating to produce sufficient product for sequencing.

MM09 – Produce a ~500bp band. The band was excised. This band size is ~275bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM10, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM10 – Produced a ~700bp band. The band was excised. This band size is ~475bp larger than the expected size of 225bp. This implies the presence of an intron in this region. This band size differs from that produced by MM09, which suggests that this primer set can be used for qPCR AND distinguish between the COX1 and COX2 isoforms.

MM11 – Produced multiple bands, of which two were excised; a ~3000bp band and a ~600bp band. These bands were excised solely based on their intensity and their immediate useability for sequencing. Will discuss with Steven on whether or not this should be repeated and the other bands excised for sequencing purposes. Both bands that were excised exceed the expected band size of ~275bp, suggesting the presence of multiple introns. Additionally, the presence of so many products suggests that the primers are not very specific, in regards to their target.

MM12 – An extremely faint band of ~350bp can be seen, however, due to it’s faintness and it’s small size (expected size was ~812bp), the band was not excised. Will discuss with Steven to see if this warrants repeating to accumulate sufficient product for sequencing purposes. No amplification of any larger products suggests the presence of introns, causing the size of the potential amplicon to exceed the capabilities of the polymerase under these cycling conditions. This is also confirmed by the MM11 PCR results in which a 3000bp band was produced. Since the primer set in MM12 has an additional 600bp at the 5′ end, this has already exceeded the abilities of the polymerase, even if this addtional 600bp does NOT include an additional intron. However, it is curious that the MM12 primer set does not produce smaller, spurious PCR products as is seen in the MM11 primer set (these two primer sets both use the same forward primer).

Genomic PCR – Repeat of C.gigas COX genomic PCR from 20110118

This was repeated to generate more PCR product for sequencing purposes. PCR master mix calcs and cycling params are here. Master mixes 04 and 05 (MM04 and MM05) were repeated to gain more PCR product from the faint 550bp & 1500bp bands(MM04) and 5000bp band (MM05).

MM04 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp

MM05 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp

Results:

Gel was run on 20110203

Samples on the left portion of the gel are from the MM04 primer combo and those on the right are from the MM05. Boxed bands were excised, purified using Millipore Ultra DA-free spin columns and stored @ -20C in Sam’s “Misc. -20C Box.”

Interestingly in the MM05 set, inconsistent, faint bands of ~400-500bp are visible. These were not visible the first time this PCR was conducted (see 20110118), but the exposure of the gel image wasn’t turned up as high as in this image. Due to their inconsistency and extremely low yield, these bands were not excised.

Genomic PCR – C.gigas cyclooxygenase (COX) genomic sequence

Attempt to obtain full genomic sequence for C.gigas COX. PCR set up/cycling params/etc are here. Primer set combinations(master mixes) are as follows:

MM01 – Cg_COX_5’UTR_3_F (SR ID: 1150) + Cg_COX_1009_R (SR ID: 1147) Band size w/o intron = ~1000bp

MM02 – “” + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~1540bp

MM03 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~2135bp

MM04 – Cg_COX_982_F (SR ID: 1151) + Cg_COX_1545_R (SR ID: 1148) Band size w/o intron = ~550bp

MM05 – “” + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~1130bp

MM06 – Cg_COX_1519_F (SR ID: 1146) + Cg_COX_2138_R (SR ID: 1149) Band size w/o intron = ~620bp

Results:

Bioline Hyperladder I used for marker. Gel is loaded with template samples at the far left of each master mix group with two no template controls (NTC) in the remaining two wells of each master mix group. All NTCs on the gel are clean.

All bands surrounded by a green box were excised from the gel.

MM01, MM02 and MM03 – The smallest expected band (i.e. no intron present) would have been 1000bp in MM01. Instead, we see faint banding of multiple sizes less than 1000bp in both MM01 and MM02. MM03 fails to produce any bands. This potentially suggests a couple of things. Firstly, the multiple banding produced in MM01 and MM02 suggests that the PCR conditions lead to some non-specific priming and should be optimized. Secondly, the fact that no bands were produced that are equal to or larger than the “no intron size” suggests that intron(s) may exist in the 5′ region of the COX gene and are large enough that the polymerase had insufficient time/ability to amplify.

MM04 – Three distinct bands were produced: 2000bp, 1500bp and 550bp. The size of band that would have been produced had an intron NOT been present would have been ~550bp. A band of this size was produced in this PCR reaction. However, two additional bands were produced. The presence of these two larger bands lends additional evidence for the existence of multiple isoforms of COX (which is also supported by the fact that multiple isoforms of COX are known to exist in most other species). The 2000bp band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

MM05 – A distinct band of ~5000bp was produced. This is significantly larger than the “no intron size” of ~1130bp, suggesting the presence of an intron. This band was excised, but not purified due to the low concentration of DNA in the gel. The gel slice was stored @ -20C until this PCR reaction could be repeated to accumulate sufficient product for sequencing.

MM06 – A distinct band of ~2200bp was produced. This is significantly larger than the “no intron size” of ~620bp, suggesting the presence of an intron. The band was excised and purified with Millipore Ultra-free DA spin columns and stored @ -20C until a sequencing plate is readied.

The PCR reactions reveal the presence of intron(s) in the COX gene we’re investigating as well as providing evidence for the existence of multiple isoforms in C.gigas. Since the PCR products that have been excised for sequencing are so large, additional primers will need to be designed closer to the introns in order to generate smaller products that can be fully sequenced. Additionally, all reactions using the primer designed to anneal in the 5’UTR of COX (SR ID: 1150) failed to produce useful results. This is either due to poor performance of the primer under these reaction conditions or due to the presence of a large intron in the 5′ region of the gene. Additional reverse primers will be designed that anneal closer to the 5′ portion of the COX gene in hopes of characterizing the 5′ genomic sequence better.

After speaking with Steven today about the potential existence/”discovery” of multiple isoforms, he decided to map the newly-released C.gigas 454 NGS data to the existing COX coding sequence in GenBank (FJ375303). The alignment is shown below.

The two 454 reads that map closest to the 5′ end of the COX coding sequence match up nearly perfectly, with periodic SNPs. The remaining 454 reads that map to the COX coding sequence are very different and provide very good evidence of a previously unidentified isoform of COX in C.gigas. Primers will be designed from both the existing COX sequence in GenBank (FJ375303) and the other potential isoform. These primers will likely be used in both qPCR and for sequencing purposes, in order to be able to distinguish and characterize both isoforms. Additionally, BLASTing will be performed with the sequences from both isoforms to evaluate how they match up with existing COX isoforms in other species.

PCR – Test Lexie’s Mercenaria 18s contamination issue

Lexie’s PCR with this primer set and a pool of Mercenria cDNA has yielded contamination in all of her waters. Performed two sets of PCR: one with her existing primer working stocks and the other with a fresh aliquot of primer working stocks. Used my own reagents/water. PCR set up and cycling params are here. PCR ran O/N.

Results:

Lane 1 – 100bp ladder

Lane 2 – H2O (Lexie’s primer stocks)

Lane 3 – H2O (Lexie’s primer stocks)

Lane 4 – cDNA (Lexie’s primer stocks)

Lane 5 – H2O (fresh primer stocks)

Lane 6 – H2O (fresh primer stocks)

Lane 7 – cDNA (fresh primer stocks)

All water samples look clean and there’s a nice bright band in the cDNA samples. Lexie’s contamination issue is probably a technique issue and not one of reagent contamination.

PCR – Sepia cDNA

This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today. Here’s yesterday’s workup

Results:

Gel Loading (from left to right):

Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)

1 – 100bp ladder

2 – retina

3 – fin

4 – 4th arm

5 – dorsal mantle center

6 – dorsal mantle side

7 – ventral mantle center

8 – ventral mantle side

9 – H2O

10 – H2O

Opsin Primers

Single bands in retina, fin and ventral mantle center samples. Negative controls are clean. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. Tubes are labelled with a “2” to differentiate them from the gel run earlier today.

Rhodopsin Primers

Single bands in retina and fin. Double bands in ventral mantle center sample. Second band is very faint is ~700bp. Negative controls are clean. Bands, including the faint 700bp VMC, will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. Tubes are labelled with a “2” to differentiate them from the gel run earlier today.

PCR – Sepia cDNA

This is an exact repeat of the PCR from yesterday, due to inconsistencies between repeated PCRs from yesterday and earlier today. Here’s yesterday’s workup. Samples were stored @ 4C O/N after completion of PCR. Gell was run on 20091217.

Results: This is getting embarrassing. Opsin results are same as yesterday (good!), but Rhodopsin results are slightly different than yesterday’s AND the day before yesterday’s results!

Gel Loading (from left to right):

Opsin Primers (lanes 2-10), Rhodopsin Primers (same loading order, lanes 12-20)

1 – 100bp ladder

2 – retina

3 – fin

4 – 4th arm

5 – dorsal mantle center

6 – dorsal mantle side

7 – ventral mantle center

8 – ventral mantle side

9 – H2O

10 – H2O

Opsin Primers

We see a band in the retina, fin, 4th arm, & ventral mantle center samples as we did yesterday. This replicates yesterday’s PCR results, which is good. Negative controls are clean. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. NOTE: This gel was run on 20091217 and the tubes are dated as such!

Rhodopsin Primers

A single band is present in the retina, dorsal mantle center and ventral mantle center samples. We see TWO bands in the fin sample. These results differ from yesterday’s in that yesterday, a single band was present ONLY in the retina and fin samples. Two days ago there was a single band in each of the retina and fin samples and two bands in the ventral mantle center sample. Bands will be excised and stored @-20C in Sam’s “Purified Inserts” box for eventual sequencing. NOTE: This gel was run on 20091217 and the tubes are dated as such!

I am going to repeat these PCRs again. I can’t stand the fact that I am getting such freaking inconsistent results in an extremely simple PCR. Aaargh!

PCR – Sepia cDNA and DNased RNA

Set up PCR on recent Sepia cDNA and DNased RNA samples using both S. officianalis_rhodopsin_F, R primers & Sep_op_F2, R2 primers. After yesterday’s intirguing PCR results, we need to confirm that the DNased RNA is free of contaminating gDNA. Additionally, we want to excise bands from the cDNA samples for sequencing. PCR set up is here. RNA was prepped as though making cDNA; diluted 0.2ug of DNased RNA in a final volume of 25uL. Used 1uL of this for PCRs.

Rhodopsin primers require 3mM Mg2+ in the PCR rxn and the opsin primers require 2mM Mg2+ in the PCR rxn. 25mM Mg2+ was added as required and is shown on the PCR set up link above.

Results: n00b alert! The gel below is a repeat of yesterday’s PCR, except with DNased RNA samples added to verify no gDNA carryover. However, you’ll notice that the PCR results on the cDNA don’t look identical to yesterday’s PCR. Ugh.

Gel Loading (from left to right):

Opsin Primers (top half of gel), Rhodopsin Primers (same loading order, bottom half of gel)

RNA samples (left sides of gel), cDNA samples (right sides of gel)

1 – 100bp ladder

2 – retina

3 – fin

4 – 4th arm

5 – dorsal mantle center

6 – dorsal mantle side

7 – ventral mantle center

8 – ventral mantle side

9 – H2O

Loading is repeated in the above order for the cDNA, which is the right halves of the gel.

Well, the good news is that no signal is seen in the DNased RNA samples, thus confirming that there is no detectable gDNA carryover.

Opsin Primers

We see a band in the retina, fin & ventral mantle center samples as we did yesterday. However, we see a band in the 4th arm sample, which was not present yesterday. We also do NOT see a band in the ventral mantle side sample like we did yesterday. Negative controls are negative.

Rhodopsin Primers

We see a band in the retina and fin samples as we did yesterday. However, we no longer see the dual bands in the ventral mantle center as were seen yesterday. Negative controls are negative.

Bands were excised from the gel, placed in 1.5mL snap cap tubes and stored in the -20C in Sam’s “Purified Inserts” box.

Because of the lack of repeated results, I will repeat this PCR for a third time, with just the cDNA again, to see if I can duplicate one set of results…