Inoculated 5mL of 1x LB + Kan50 (made by Steven on 3/23/11). Incubated O/N @ 37C, 250RPM. Will perform mini preps tomorrow. The following samples were selected (red text on the plate):
- #9
- #10
- #13
- #43
- #45
- #49
- #56
- #58
Tag Archives: COX
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:
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.
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…
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.
Ligations – COX1/COX2 PCR Products
Performed ligations/cloning on a variety of COX1 genomic and COX 5′ RACE products using the TOPO TA Cloning Kit (Invitrogen). Used 2uL of gel-purified PCR product in each cloning rxn, 2.5uL of H2O, 1uL of salt solution, and 0.5uL of the Invitrogen pCR2.1 vector. Incubated samples for 5mins at RT. Used 2uL of the cloning reaction to transform TOP10 chemically competent cells (Invitrogen), mixed very gently, incubated on ice for 5mins, heat shocked at 42C for 30s and immediately placed on ice. Added 250uL of SOC Medium and incubated tubes at 37C, 200RPM for 1hr. Plated 50uL of each transformation on LB+Kan plates (made by Steven on unknown date with unknown Kan concentration) containing 40uL of 40mg/mL X-gal. Incubated O/N at 37C. Remaining volume of transformed bacteria were stored @ 4C.
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.
qPCR – C.gigas COX1/COX2 Tissue Distribution
Performed qPCR using pooled cDNA from 20110311. Pooled 2uL from each of the following samples groups: Dg 3hr C, Gill 1hr C, Gill 1hr E, Mantle 3hr C, and Muscle 3hr C. Master mix calcs are here. Plate layout, cycling params, etc can be found in the qPCR Report (see Results). Primers sets run were:
EF1_qPCR_5′,3′ (SR IDs: 309, 310)
Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX1_qPCR_R (SR ID: 1191)- Target = COX1
Cg_COX1/2_qPCR_F (SR ID: 1192) + Cg_COX2_454align1_R (SR ID: 1190) – Target = COX2
Results:
Graphs were generated using the BioRad CFX Manager v2.0 software. Expression was normalized to EF1. Also to note, gene efficiency was assumed as 100% by the software since no standard curve was run on the plate. As such, analysis of this data may not be exact.
It’s clear by examining the graphs that the primers being used to differentiate COX1 and COX2 (since they share a common primer: SRID 1192) are differentially expressed. This indicates that the primer sets are indeed amplifying different targets as hoped. This was the primary intention of this qPCR. However, we also now have an idea of tissue distribution of the two genes, as well as their response to V. vulnificus exposre after 1hr. Next step is to perform this qPCR on all the individuals from this experiment as well as the different tissues.
3’RACE – C.gigas 3’RACE for COX2
Used Cg_COX2_3’RACE_short (SR ID: 1197) & Cg_COX2_3’RACE_long (SR ID: 1196) and the Clonetech SMART RACE cDNA Amplification Kit (unknown acquisition date) to attempt to acquire more 3′ sequence of the C.gigas COX2 isoform. Used Gigas 3’RACE cDNA (from 20080610).
Results:
Gel Loading:
Lane 1: Hyperladder 1
Lane 2: empty
Lane 3: Cg_COX2_3’RACE_long
Lane 4: Cg_COX2_3’RACE_long NTC
Lane 5: empty
Lane 6: Cg_COX2_3’RACE_short
Lane 7: Cg_COX2_3’RACE_short NTC
Lane 8: Hyperladder 1
No products produced. This could be due to a large number of factors. The age of the cDNA (from 20080610) is well beyond what the Clontech manual says for storage term (6 months). Additionally, the Clontech polymerase used was nearly 6 years old. The kit (and its components) are of an unknown age and could factor in to the failure of this procedure. Also, the primers that were designed had less than ideal Tm, per the kit’s recommendations.
May need to sequence some previously purified potential COX2 fragments in order to obtain a more useable region of the gene for RACE.